A novel pectin from Akebia trifoliata var. australis fruit peel and its use as a wall-material to coat curcumin-loaded zein nanoparticle.

In this study, a pectin was extracted from Akebia trifoliata var. australis fruit peel waste using water solution, and its physicochemical properties were evaluated. The pectin was rich in galacturonic acid (GalA) content (76.68%). The degree of esterification (DE) and molecular weight (Mw) were 37.60% and 29,890 Da, respectively. The pectin structure was determined using Fourier transform-infrared (FT-IR) and Hydrogen nuclear magnetic resonance (H-NMR). The pectin exhibited an amorphous nature, negative charge, and good solubility. The pectin was then used as a wall-material to coat curcumin-loaded zein nanoparticles for the first time. The obtained nanoparticles (curcumin-loaded core-shell nanoparticle, CLCSNs) exhibited a core (zein)-shell (pectin) structure and a spherical shape with an average diameter of 230 nm. The electrostatic attraction, hydrogen bonding, and intermolecular interaction were involved in the CLCSNs formation. A high encapsulation efficiency (EE, 89.65%) and loading capacity (LC, 10.35%) of the CLCSNs were obtained for the curcumin. The solubility, stability, antioxidant activity, and in vitro bioavailability of the curcumin were significantly increased after loading into the CLCSNs. Therefore, this sustainable pectin from Akebia trifoliata var. australis fruit peel waste represents a promising natural macromolecule for use in the pharmaceutical and food industries.

The Krüppel-associated box repressor domain induces reversible and irreversible regulation of endogenous mouse genes by mediating different chromatin states.

The Krüppel-associated box (KRAB) domain is a transcription repression module from the largest family of transcriptional regulators encoded by higher vertebrates. We developed a drug-controllable regulation system based on an artificial KRAB-containing repressor (tTS) that targets the endogenous Hprt gene to explore the regulatory mechanism and molecular basis of KRAB-containing regulators within the context of an endogenous gene in vivo. We show that KRAB can mediate irreversible and reversible regulation of endogenous genes in mouse that is dependent on embryonic developmental stage. KRAB-induced stable DNA methylation within the KRAB binding region during the early embryonic stage, resulting in irreversible gene repression. In later stages, KRAB mainly induced de-acetylation and methylation of histone, resulting in reversible gene repression. Thus, we have characterized the KRAB-mediated regulation system within the context of an endogenous gene and multiple spatiotemporal ranges, thereby providing a basis for identifying the function of KRAB-containing regulators and aiding development of novel KRAB-based gene regulation tools in vivo.

Inducible and reversible regulation of endogenous gene in mouse.

Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene's promoter region turns the gene on and off in a tetracycline-inducible manner. Nmyc(TRE) mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. Nmyc(TRE) mice were crossed to tTS transgenic mice to produce Nmyc(TRE/TRE): tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS-dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed.

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function.

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.

The stimulation of HSD17B7 expression by estradiol provides a powerful feed-forward mechanism for estradiol biosynthesis in breast cancer cells.

Our laboratory has previously cloned and purified an ovarian protein found to be a novel 17ß-hydroxysteroid dehydrogenase type 7 enzyme (HSD17B7) (formerly prolactin receptor-associated protein) that converts the weak estrogen, estrone, to the highly potent estradiol. The regulation of this enzyme has not yet been explored. In this report, we show high expression of HSD17B7 in human ductal carcinoma and breast cancer cell lines and present evidence for a strong up-regulation of this enzyme by estradiol at the level of mRNA, protein expression, and promoter activity in MCF-7 cells. The effect of estradiol is mediated by estrogen receptor (ER)α, whereas ERß prevents this stimulation. ER antagonists, ICI 182,780 and 4-hydroxytamoxifen, prevent estradiol-induced stimulation of the endogenously expressed HSD17B7, suggesting that these inhibitors not only block estradiol action but also its production. We have identified a -185-bp region of the hsd17b7 promoter that is highly conserved among rat, mouse, and human and confers regulation by estradiol in MCF-7 cells. This region is devoid of a classical estradiol-response element but contains a nuclear factor 1 (NF1) site that is essential for estradiol action. We found that estradiol stimulates the recruitment and DNA binding of NF1 to this region of the hsd17b7 promoter. Furthermore, knockdown of NF1 family members, NF1B, NF1A, and NF1X, completely prevents induction of this gene by estradiol. In summary, our findings demonstrate that estradiol stimulates HSD17B7 transcriptional activity in breast cancer cells through a novel mechanism requiring NF1 and strongly suggest a positive feedback mechanism to increase local estradiol synthesis causing growth of estrogen-dependent breast cancers.

Prolactin receptor-associated protein/17beta-hydroxysteroid dehydrogenase type 7 gene (Hsd17b7) plays a crucial role in embryonic development and fetal survival.

Our laboratory has previously cloned and purified a protein named PRAP (prolactin receptor-associated protein) that was shown to be a novel 17beta-hydroxysteroid dehydrogenase (HSD) enzyme with dual activity. This enzyme, renamed HSD17B7 or PRAP/17beta-HSD7, converts estrone to estradiol and is also involved in cholesterol biosynthesis. The major site of its expression is the corpus luteum of a great number of species including rodents and humans. To examine the functional significance of HSD17B7 in pregnancy, we generated a knockout mouse model with targeted deletions of exons 1-4 of this gene. We anticipated a mouse with a severe fertility defect due to its inability to regulate estrogen levels during pregnancy. The heterozygous mutant mice are normal in their development and gross anatomy. The females cycle normally, and both male and female are fertile with normal litter size. To our surprise, the breeding of heterozygous mice yielded no viable HSD17B7 null mice. However, we found HSD17B7 null embryo alive in utero on d 8.5 and d 9.5. By d 10.5, the fetuses grow and suffer from severe brain malformation and heart defect. Because the brain depends on in situ cholesterol biosynthesis for its development beginning at d 10, the major cause of fetal death appears to be due to the cholesterol synthetic activity of this enzyme. By ablating HSD17B7 function, we have uncovered, in vivo, an important requirement for this enzyme during fetal development.

Involvement of cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) in interleukin 11 stimulation of decidualization in mice.

Interleukin 11 receptor alpha (Il11ra) null mice are infertile due to defective decidualization and abnormal trophoblast invasion. We have previously shown in these mice that downregulation of decidual proteinase inhibitors plays a role in uncontrolled trophoblast invasion. However, the decidua is abnormally smaller in pseudopregnant Il11ra null mice, where trophoblast invasion is not a factor. Here, we examined whether defective decidualization is due to dysregulation of key molecules involved in decidual cell growth and differentiation. We found a dramatic downregulation of cyclin D3 in Il11ra null mice. We also found that IL11 robustly stimulates the expression of cyclin D3 in cell culture. CDK4 and CDK6, known partners of cyclin D3, are not affected. Immunolocalization studies show absence of cyclin D3 in the mesometrial site and absence of differentiated polyploid cells in the antimesometrial site of Il11ra null mice. We also examined the expression of cell differentiation factors CDKN1A (p21) and CDKN1B (p27), and found that in both in vivo and cell culture the expression of CDKN1A (p21) but not CDKN1B (p27) is under the control of IL11. Another clear target of IL11 in the decidua is BIRC5 (Survivin), whose expression is repressed in the decidua of Il11ra null mice and stimulated by IL11 in cell culture. Taken together, these results provide, at least in part, an explanation for the defective small decidua of mice lacking the Il11ra gene, and reveal for the first time that cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) are targets of IL11 in the decidua.

Inhibition of GSK-3beta decreases NF-kappaB-dependent gene expression and impairs the rat liver regeneration.

Serine-threonine protein kinase glycogen synthase kinase (GSK)-3 is involved in regulation of many cell functions, but its role in regulating liver regeneration is unknown. Here we investigated the effects of GSK-3beta inhibition on liver regeneration after partial hepatectomy in the rat. The potent and selective GSK-3beta inhibitor SB216763 (0.6 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered 30 min before 70% partial hepatectomy. Liver regeneration was estimated by the cell proliferation, apoptosis, and the related cell signaling and cycling proteins. In 30 min after hepatectomy in the rat, GSK-3beta was found to be translocated to the nucleus, but GSK-3beta inhibitor SB216763 that could phosphorylate residue Ser9 on GSK-3beta did not attenuated the accumulation. Consequently, the inhibition of GSK-3beta decreased the nuclear factor-kappaB activity, the NF-kappaB-dependent gene expression, and COX2 expression, but enhanced p21(WAF1/Cip1) transcription. Moreover, the injection of SB216763 impaired the proliferation cell nuclear antigen (PCNA) index and increased the apoptosis of liver compared to the vehicle. GSK-3beta plays an important role in rat liver regeneration. We conclude it may partially result from the inhibition of the NF-kappaB pathway and enhancement of p21 (WAF1/Cip1) expression.

[Adenoviral-mediated expression of PTEN cDNA induces apoptosis in endometrial carcinoma cells].

OBJECTIVE: To investigate whether the human endometrial carcinoma cells, RL95-2 infected with recombinant Ad-PTEN can steadily produce PTEN protein and enter apoptosis. METHODS: The recombinant adenovirus containing PTEN cDNA was constructed using the method of homologous recombination in bacteria. The viral titer was examined by plaque assay and the expression of PTEN protein was detected by western blot assay. The apoptosis of RL95-2 cells was evaluated as following: flipping of membrane phosphatidylserine (PS) and identification of activating caspase-3 positive cells was determined by flow cytometer (FCM), and furthermore genomic DNA fragmentation was detected by agarose electrophoresis. RESULTS: The recombinant adenovirus encoding PTEN cDNA was successfully constructed, and viral titers of Ad-PTEN were 5 x 10(9) pfu/ml. After infected by Ad-PTEN, the expression of PTEN protein was steady in human RL95-2 cells. After infected by Ad-PTEN for 24, 48, 72 and 96 h, the relative cell number of membrane PS flipping were (6.09 +/- 1.01)%, (9.98 +/- 2.17)%, (11.74 +/- 2.65)%, (27.69 +/- 8.67)%, which significantly increased than control group (P < 0.05), the relative cell number of activated caspase-3 positive were (2.6 +/- 0.5)%, (18.0 +/- 4.4)%, (21.8 +/- 5.1)%, (33.7 +/- 9.9)%, respectively, which significantly increased than control group (P < 0.05), and genomic DNA fragmentation was verified also. CONCLUSIONS: The recombinant Ad-PTEN vector is constructed successfully and the expression of specific PTEN is steady in RL95-2 cell line. The expression of PTEN induces RL95-2 cells to apoptosis. PTEN gene may be a novel therapeutic target in endometrial carcinoma.

Cloning and characterization of a 5' regulatory region of the prolactin receptor-associated protein/17{beta} hydroxysteroid dehydrogenase 7 gene.

Prolactin receptor-associated protein (PRAP) originally cloned in our laboratory was shown to be a novel, luteal isoform of 17beta hydroxysteroid dehydrogenase 7 (17betaHSD7). In this study, we cloned the promoter region of rat PRAP/17betaHSD7 and investigated the mechanisms regulating both basal activity and LH-induced repression of this promoter. Truncated and site-specific mutants of PRAP/17betaHSD7 promoter identified two enhancer regions that contained highly conserved Sp1 binding site and bound Sp1 from nuclear extracts of both corpora lutea and a rat luteal cell line. Repression of PRAP/17betaHSD7 expression and promoter activity by human chorionic gonadotropin/forskolin was localized to a -52-bp proximal segment of the promoter. This region contained a conserved CCAAT site and bound nuclear factor Y; binding of this transcription factor was inhibited by human chorionic gonadotropin in vivo. Furthermore, mutation of the nuclear factor Y site in the -52-bp promoter-reporter construct abolished forskolin-mediated inhibition of the promoter in a rat luteal cell line. In summary, we have identified the promoter elements involved in the basal expression of PRAP/17betaHSD7. We have also found that LH-mediated repression of this gene is at the level of transcription and involves inhibition of nuclear factor YA binding to the CCAAT site within the proximal promoter.

Study on intracellular trafficking of Mac-1 by direct visualization.

Previously, we constructed DNA vectors containing cDNA of Mac-1 subunits (CD11b or CD18b) fused with fluorescence protein (FP). cDNA fragments and the DNA constructs were then transfected into CHO cells (as CHO-Mac-1-FP). The structure and function of Mac-1-FP obtained from the CHO-Mac-1-FP cells are nearly identical to that expressed in wild type leukocytes. In the present study, the intracellular trafficking of Mac-1 was visualized directly by monitoring the fluorescent intensities of YFP-CD18 and PE-conjugated monoclonal antibody against CD11b under a confocal microscope in CHO-Mac-1-FP cells. The results indicate that: (i) although Mac-1 was not detected in the cell membrane at resting state, it had been translocated and clustered into the cell membrane by 1 h and internalized 2 h after PMA stimulation, at which point the fluorescence intensity began to diminish gradually, probably due to partial degradation of Mac-1. The fluorescence of CD18 and CD11b reappeared on the cell membrane 1 h after re-treatment with PMA, suggesting the recycling of non-degraded Mac-1. (ii) The adhesion rate of CHO-Mac-1-FP to magnetic beads coupled ICAM-1 increased within 4 h after their initial interaction, accompanied by the clustering of Mac-1-FP. After 8 h, the adhesion rate declined and fluorescence also decreased simultaneously. The pattern of change in fluorescence in CHO-Mac-1-FP cells elicited by ICAM-1 beads was similar to that elicited by PMA, suggesting that endocytosis and degradation of Mac-1 occurred after the interaction with ICAM-1. Thus, we conclude that the intracellular trafficking of Mac-1 after activation is associated with membrane translocation, endocytosis, degradation and recycling. These changes are in parallel with the adhesion of CHO-Mac-1-FP cells with ICAM-1, and may be involved in the adhesion and detachment of leukocytes. The detachment of leukocytes may be caused by endocytosis of Mac-1.

Expression, purification, and C-terminal amidation of recombinant human glucagon-like peptide-1.

Human glucagon-like peptide-1 (hGLP-1) (7-36) amide, a gastrointestinal hormone with a pharmaceutical potential in treating type 2 diabetes mellitus, is composed of 30 amino acid residues as a mature protein. We report here the development of a method for high-level expression and purification of recombinant hGLP-1 (7-36) amide (rhGLP-1) through glutathione S-transferase (GST) fusion expression system. The cDNA of hGLP-1-Leu, the 31st-residue leucine-extended precursor peptide, was prepared by annealing and ligating of artificially synthetic oligonucleotide fragments, inserted into pBluescript SK (+/-) plasmid, and then cloned into pGEX-4T-3 GST fusion vector. The fusion protein GST-hGLP-1-Leu, expressed in Escherichia coli strain BL21 (DE3), was purified by affinity chromatography after high-level culture and sonication of bacteria. Following cleavage of GST-hGLP-1-Leu by cyanogen bromide, the recombinant hGLP-1-Leu was released from fusion protein, and purified using QAE Sepharose ion exchange and RP C(18) chromatography. After purification, the precursor hGLP-1-Leu was transacylated by carboxypeptidase Y, Arg-NH(2) as a nucleophile, to produce rhGLP-1. Electrospray ionization mass spectrometry showed the molecular weight was as expected. The biological activity of rhGLP-1 in a rat model demonstrated that plasma glucose concentrations were significantly lower and insulin concentrations higher after intraperitoneal injection of rhGLP-1 together with glucose compared with glucose alone (P < 0.001).

Solution structure and biological activity of recombinant salmon calcitonin S-sulfonated analog.

Salmon calcitonin S-sulfonated analog (abbreviated as [S-SO(3)(-)]rsCT) was prepared by introducing two sulfonic groups into the side chains of Cys1 and Cys7 of recombinant salmon calcitonin. The hypocalcemic potency of this open-chain analog is 5500IU/mg, which is about 30% higher than that (4500IU/mg) of the wild type. The solution conformation of [S-SO(3)(-)]rsCT was studied in aqueous trifluoroethanol solution by CD, 2D-NMR spectroscopy, and distance geometry calculations. In the mixture of 60% TFE and 40% water, the peptide assumes an amphipathic alpha-helix in the region of residues 4-22, which is one turn longer than that of the native sCT. The structural feature analysis of the peptide revealed the presence of hydrophobic surface composed of five hydrophobic side chains of residues Leu4, Leu9, Leu12, Leu16, and Leu19, and a network of salt-bridges that consisted of a tetrad of oppositely charged side chains (Cys7-SO(3)(-)-Lys11(+)-Glu15(-)-Lys18(+)). The multiple salt bridges resulted in the stabilization of the longer amphipathic alpha-helix. Meanwhile, the higher hypocalcemic potency of the peptide could be attributed to the array of hydrophobic side chains of five leucine residues of the amphipathic alpha-helix.

Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring.

Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.

Purification, crystallization and preliminary X-ray studies of human augmenter of liver regeneration.

Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A. Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7).