Therapeutic potential of amniotic fluid stem cells to treat bilateral ovarian dystrophy in dairy cows in a subtropical region.

Amniotic fluid is a rich source of multipotent mesenchymal stem cells (MSCs). Amniotic fluid stem cells (AFSCs) have become a new source of stem cells; they have low immunogenicity and are easily harvested. For this reason, they may be useful in clinical tissue engineering. Moreover, AFSCs have anti-inflammatory properties and can repair tissues. This study evaluated the utility of AFSC injection to treat bilateral ovarian dystrophy in Holstein-Friesian cows. Bovine AFSCs (BAFSCs) were collected at slaughter from Holstein-Friesian cows during the third or fourth month of pregnancy and cultured in vitro. The BAFSCs began to show a fibroblast-like morphology. They were positive for ß-integrin, CD44, CD73, CD106 and Oct4 and negative for CD34 and CD45. After induction, the cells differentiated into mesodermal lineages. Bilateral ovarian dystrophy was confirmed by ultrasonography in 16 lactating cows. The subsequent experiment lasted 15 weeks. Serum was collected weekly to analyse progesterone concentrations, and weekly ultrasonography recorded ovarian changes. Each cow was equipped with an automatic heat detection system to facilitate oestrus observation and breeding records. The progesterone concentration of two cows in the treatment group (25%) significantly increased during weeks 10-15. On ultrasonography, the treatment group demonstrated mature follicles after BAFSCs injection, and foetuses were visualized approximately 40 days after artificial insemination (AI). Oestrus rates in the control and treatment groups were 0% (0/8) and 50% (4/8), respectively; pregnancy rates were 0% (0/8) and 25% (2/8), respectively. Calves were successfully delivered in both cases of pregnancy. These results show that BAFSCs can alleviate bovine ovarian dystrophy and restore fertility.

Tunable PEGylation of branch-type PEI/DNA polyplexes with a compromise of low cytotoxicity and high transgene expression: in vitro and in vivo gene delivery.

Although PEGylated polyplexes for gene delivery are widespread, there is a need for an in-depth investigation of the role of the PEGylation degree on the delivery efficiency of the systems. For this, a low-toxicity series of polymers for gene delivery were designed via Michael addition of poly(ethylene glycol)methyl ether methacrylate (PEGMA) onto branched polyethylenimine PEI. The goal was to finely tune the PEGylation degree in order to determine the system offering the best compromise between low cytotoxicity and high transfection efficiency under both in vitro and in vivo conditions. From dynamic light scattering tests, zeta potential measurements and gel retardation assay, it was found that nanoparticle assembly of PEI-g-PEGMA and DNA exhibited stable complex formation when the PEGylation degree was below 2.9%. In addition, complexes formed from polymers with a PEGylation degree of at least 1.67% (from PEI-g-PEGMA-6 to PEI-g-PEGMA-18) all showed very low hemolysis activity. Transfection efficiencies of the prepared complexes were determined using the pEGFP-C3 vector and ß-galactosidase. Complexes made of PEI-g-PEGMA-6 and PEI-g-PEGMA-10 at a polymer nitrogen/DNA phosphorus weight ratio (Wn/Wp) of 5 led to the best transfection efficiencies. Moreover, PEGylation ensured low cytotoxicity of the complexes in particular at high Wn/Wp ratios. In vivo tests in a mouse model confirmed the in vitro results obtained for PEI-g-PEGMA-6-based complexes, at all Wn/Wp ratios tested, but also showed that a high PEGylation degree (5.2% for PEI-g-PEGMA-18), though inefficient in vitro could still lead to successful delivery in vivo, due to a prolonged contact time between the complex and the cells, and to the change in the biological environment. Overall, provided a fine tuning of the grafting density of PEGMA onto PEI and the polymer nitrogen/DNA phosphorus weight ratio, our results prove that PEI-g-PEGMA polymers constitute an efficient platform for successful in vitro and in vivo gene delivery, and ensure low cytotoxicity and prolonged cell viability.

Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2.

Novel Vitamin K analogs suppress seizures in zebrafish and mouse models of epilepsy.

Epilepsy is a debilitating disease affecting 1-2% of the world's population. Despite this high prevalence, 30% of patients suffering from epilepsy are not successfully managed by current medication suggesting a critical need for new anti-epileptic drugs (AEDs). In an effort to discover new therapeutics for the management of epilepsy, we began our study by screening drugs that, like some currently used AEDs, inhibit histone deacetylases (HDACs) using a well-established larval zebrafish model. In this model, 7-day post fertilization (dpf) larvae are treated with the widely used seizure-inducing compound pentylenetetrazol (PTZ) which stimulates a rapid increase in swimming behavior previously determined to be a measurable manifestation of seizures. In our first screen, we tested a number of different HDAC inhibitors and found that one, 2-benzamido-1 4-naphthoquinone (NQN1), significantly decreased swim activity to levels equal to that of valproic acid, 2-n-propylpentanoic acid (VPA). We continued to screen structurally related compounds including Vitamin K3 (VK3) and a number of novel Vitamin K (VK) analogs. We found that VK3 was a robust inhibitor of the PTZ-induced swim activity, as were several of our novel compounds. Three of these compounds were subsequently tested on mouse seizure models at the National Institute of Neurological Disorders and Stroke (NINDS) Anticonvulsant Screening Program. Compound 2h reduced seizures particularly well in the minimal clonic seizure (6Hz) and corneal-kindled mouse models of epilepsy, with no observable toxicity. As VK3 affects mitochondrial function, we tested the effects of our compounds on mitochondrial respiration and ATP production in a mouse hippocampal cell line. We demonstrate that these compounds affect ATP metabolism and increase total cellular ATP. Our data indicate the potential utility of these and other VK analogs for the prevention of seizures and suggest the potential mechanism for this protection may lie in the ability of these compounds to affect energy production.

Isolation and characterization of an active compound from black soybean [Glycine max (L.) Merr.] and its effect on proliferation and differentiation of human leukemic U937 cells.

Black soybean [Glycine max (L.) Merr.] has been used as a health food and herb in China for hundreds of years. In the present study, we purified a unique polysaccharide component from black soybean (PSBS) and found that it indirectly inhibits proliferation and induces differentiation of human leukemic U937 cells via activation of mononuclear cells (MNCs). We prepared conditioned media (MNC-CM) by incubating MNCs from human peripheral blood with or without PSBS (PSBS-MNC-CM and normal MNC-CM, respectively). Treatment of human leukemic U937 cells with PSBS-MNC-CM significantly inhibited proliferation of U937 cells, reducing their growth by 98.5%. Furthermore, PSBS-MNC-CM induced U937 cells to differentiate into mature monocytes/macrophages (83% by morphological examination and 90% by the nitroblue tetrazolium test). Neither PSBS alone nor normal MNC-CM had such effects. The molecular weight of PSBS was about 480 000 by gel filtration. Structural analysis of PSBS revealed that (1,6)-alpha-D-glucan might be its major active component. Our results suggest that the PSBS may inhibit proliferation and induce differentiation in human leukemic U937 cells by activating the immune response of MNCs.

C. elegans CED-12 acts in the conserved crkII/DOCK180/Rac pathway to control cell migration and cell corpse engulfment.

We have identified and characterized a novel C. elegans gene, ced-12, that functions in the conserved GTPase signaling pathway mediated by CED-2/Crkll, CED-5/DOCK180, and CED-10/Rac to control cell migration and phagocytosis of apoptotic cells. We provide evidence that ced-12 likely acts upstream of ced-10 during cell migration and phagocytosis and that CED-12 physically interacts with CED-5 and forms a ternary complex with CED-2 in vitro. We propose that the formation and localization of a CED-2-CED-5-CED-12 ternary complex to the plasma membrane activates CED-10, leading to the cytoskeletal reorganization that occurs in the polarized extension of cell surfaces in engulfing cells and migrating cells. We suggest that CED-12 counterparts in higher organisms regulate cytoskeleton dynamics, as CED-12 does in C. elegans.

Baicalein induces a dual growth arrest by modulating multiple cell cycle regulatory molecules.

Baicalein, a flavonoid present in the root of Scutellaria baicalensis Georgi, has been reported to inhibit cell proliferation in several types of cells. In this study, the effect of baicalein on cell growth and the mechanism of growth modulation were examined in primary cultured rat heart endothelial cells. Here, we report that treatment with 100-microM baicalein caused an almost complete inhibition of cell proliferation after 5 days of incubation. Baicalein mediated G1 and G2 growth arrest accompanied by the down-regulation of cyclin D2, cyclin A, cyclin-dependent kinase 1 (Cdk1) and cyclin-dependent kinase 2 (Cdk2), and up-regulation of p15(Ink4B), p21(CIP1/Waf1), p53 and cyclin E. Evaluation of the kinase activity of cyclin-Cdk complexes showed that baicalein decreased Cdk1, Cdk2, cyclin D2 and cyclin A expression in endothelial cells, leading to markedly reduced Cdk/cyclin-associated kinase activities. These results suggest that baicalein inhibits the proliferation of rat heart endothelial cells via G1 and G2 arrest in association with the down-regulation of the expression and function of Cdk1, Cdk2, cyclin D2 and cyclin A proteins, and up-regulation of cyclin E, p15(Ink4B), p53 and p21(CIP1/Waf1).

Camptothecin suppresses nitric oxide biosynthesis in RAW 264.7 macrophages.

Nitric oxide is an important cellular mediator that plays a role in tumor growth and angiogenesis. The present study was conducted to evaluate whether camptothecin (CPT), a topoisomerase I inhibitor, exhibits antitumor activity through regulation the inducible nitric oxide synthase (iNOS) biosynthesis pathway. Experiment was performed on RAW 264.7 cells, a transformed macrophage-like cell line, stimulated with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Incubation of RAW264.7 cells with CPT (0.1 to 10 microM) inhibited the LPS/IFN-gamma-induced nitrite accumulation in a concentration-dependent manner with an IC50 value of 0.59+/-0.07 microM. Treatment of cells with concentrations of CPT (< or =3 microM) that are not growth inhibitory or cytotoxic strongly inhibited their ability to express iNOS mRNA and iNOS protein, however, without a direct regulatory effect on iNOS activity. Time course analysis also revealed that CPT acted in a fashion similar to the transcription inhibitor actinomycin-D. Thus, the suppressant effects of CPT on LPS/IFN-gamma-stimulated NO production seemed to be mediated probably through inhibition of iNOS gene transcription. From this observation we propose that inhibition of NO biosynthesis by CPT may underlie, at least in part, the efficacy of this antitumor agent.

Cytotoxic principles from Ventilago leiocarpa.

Three new anthraquinones, islandicin 4-methyl ether (1), 1,2,6-trihydroxy-7,8-dimethoxy-3-methylanthraquinone (2), and 2-hydroxyemodin 1-methyl ether (3) as well as two known triterpenoids [taraxerol (4), lupeol (5)], six anthraquinones [chrysophanol (6), islandicin (8), parietin (9), emodin (10), catenarin (11), skyrin (15)], a 2,3-dihydroflavonol [(+)-aromadendrin (12)], two benzisochromanquinones [ventiloquinone K (13) and ventiloquinone I (14)], and stigmasterol (7) were isolated from Ventilago leiocarpa. The cytotoxicity of these compounds to various tumor cell lines was evaluated, and compound 15 significantly suppressed growth of HeLa, Vero, K562, Raji, Wish, and Calu-1 tumor cell lines. With the exception of K562 cells, the proliferation of other tumor cell lines was inhibited by compounds 3 and 10.

Regulation of cell proliferation, gene expression, production of cytokines, and cell cycle progression in primary human T lymphocytes by piperlactam S isolated from Piper kadsura.

Effects of piperlactam S (C(17)H(13)NO(4); mol. wt. 295) isolated from Piper kadsura on phytohemagglutinin (PHA) stimulated cell proliferation were studied in primary culture of human T cells. The results showed that piperlactam S suppressed T cell proliferation at about 0 to 12 h after stimulation with PHA. Synthesis of total cellular proteins and RNA in activated cell cultures was also suppressed. The inhibitory action of piperlactam S was not through direct cytotoxicity. Cell cycle analysis indicated that piperlactam S arrested the cell cycle progression of activated T cells from the G(1) transition to the S phase. In an attempt to further localize the point in the cell cycle at which arrest occurred, a set of key regulatory events leading to the G(1)/S boundary, including gene expression of cytokines and c-Fos protein synthesis, was examined. Piperlactam S suppressed, in activated T lymphocytes, the production and mRNA expression of cytokines such as interleukin-2 (IL-2), IL-4, and interferon-gamma in a dose-dependent manner. In addition, Western blot analysis indicated that c-Fos protein expressed in activated T lymphocytes was decreased by piperlactam S. Results of kinetic study indicated that inhibitory effects of piperlactam S on IL-2 mRNA expressed in T cells might be related to blocking c-Fos protein synthesis. Thus, the suppressant effects of piperlactam S on proliferation of T cells activated by PHA seemed to be mediated, at least in part, through inhibition of early transcripts of T cells, especially those of important cytokines, IL-2, IL-4, and arresting cell cycle progression in the cells.

Cytotoxic biflavonoids from Selaginella delicatula.

Four new biflavonoids-robustaflavone 4'-methyl ether (1), robustaflavone 7,4'-dimethyl ether (2), 2",3" -dihydrorobustaflavone 7,4', -dimethyl ether (3), and 2",3" '-dihydrorobustaflavone 7,4', 7"-trimethyl ether (4)-as well as two known biflavonoids, robustaflavone and amentoflavone, and three caffeoylquinic acids, 3,5-di-O-caffeoylquinic acid, 3, 4-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, were isolated from Selaginella delicatula. The structures of the new compounds were established by spectroscopic analysis and chemical modification. The cytotoxic activity of these compounds on various tumor cell lines was evaluated, and both compounds 1 and 3 significantly suppressed the growth of Raji and Calu-1 tumor cell lines.

Protein constituent contributes to the hypotensive and vasorelaxant activities of Cordyceps sinensis.

Cordyceps sinensis is a herb medicine in China for the treatment of general debility after sickness and for persons of advanced age. In the present study, cordyceps sinensis was extract by phosphate buffer saline (PBS) and dialyzed overnight against PBS using a membrane cut off at 3,500 dalton molecular weight. The resulting macromolecule fraction (defined as CS) was assayed in anesthetized rats for hypotensive effects and in isolated aorta for vasorelaxant effects. Intravenous injection of CS (8,16, 24 and 32 mg/kg, respectively) suppressed significantly the mean arterial pressure (MAP) in a dose-dependent manner. 32 mg/kg of CS induces the maximal hypotensive response with a 58 +/- 4 mm Hg (from 107 +/- 6 to 49 +/- 3 mm Hg) change in MAP and a over 45 min action duration. In aortic rings precontracted with phenylephrine treatment with CS between 0.5 and 500 microg/ml induced dose dependent relaxation. Maximal vasorelaxant response evoked by 150 microg/ml CS was 68.9 +/- 7.3%. Furthermore, CS-induced vasorelaxation is mediated by the endothelium possibly by stimulating the release of the nitric oxide and endothelium-derived hyperpolarizing factor. In conclusion, the present study revealed that presence of a constituent in CS which reduces MAP by relaxing the vascular beds directly. However, the effect may be caused by a single active ingredient or by the combined action of many active agents found in the extract.

Effects of methanol extract of Chansu on hypothalamic-pituitary-testis function in rats.

Chansu, a galenical preparation of the dried white venom of Chinese Bufo bufo gargarizans, is one of the major components of Kyushin, a traditional Chinese medicine. Kyushin is reported to have a cardiotonic effect that has been suggested to be due to the action of bufadienolides such as bufalin and cinobufagin. Recently, we found that administration of bufalin in male rats diminished the luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) and the secretion of testosterone both in vivo and in vitro. These observations suggest that Chansu may possess hypogonadal effects in male rats. In the present study, the effects of the methanol extract of Chansu on hypothalamic-pituitary-testicular function in male rats were examined. Crude Chansu was extracted by methanol and purified by a Sep-Pak C18 column. No activity of bufalin, cinobufagin, estradiol, or digoxin in purified methanol extract was detected; all Chansu used in this study was the purified methanol extract. A single intravenous injection of Chansu resulted in a decrease of the basal (20% to 55%) and human chorionic gonadotropin (hCG)-induced (35% to 40%) levels of plasma testosterone and the GnRH-induced level of plasma LH (25% to 30%). Administration of Chansu in vitro decreased basal and hCG-stimulated testosterone production by 60% to 70% and 40% to 60%, respectively, as well as spontaneous and forskolin- or 3-isobutyl-1-methylxanthine (IBMX)-induced accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) by 30% to 45% in rat testicular interstitial cells. Although LH release by rat anterior pituitary glands was diminished, GnRH release by the rat mediobasal hypothalamus was enhanced by administration of Chansu in vitro. These results suggest that the bufalin-free extracts of Chansu inhibit testosterone secretion in rats, in part, due to (1) a decreased production of testicular cAMP, (2) a decreased response of testosterone to gonadotropin, and (3) a reduction of the LH response to GnRH.

Cytotoxic constituents from the stems of Diospyros maritima.

One novel coumaric acid ester of lupeol, dioslupecin A (1), three naphthoquinones, 8'-hydroxyisodiospyrin (2), isodiospyrin (3), and plumbagin (4), three triterpenes, lupeol, lupenone and taraxerone, and four sterols, beta-sitosterol, stigmasterol, stigmast-4-en-3-one and ergosta-4,6,8(14),22-tetraen-3-one were isolated from the n-hexane extract of the stems of Diospyros maritima Blume. The structural determination of 1 was based on 1D and 2D NMR spectra (including 1H-1H COSY, 1H-13C COSY, and HMBC). All compounds were evaluated for in vitro cytotoxicity in 4 cancer cell lines. Compound 2 showed similar cytotoxicity against hepatoma (HEPA-3B, ED50 = 1.72 micrograms/ml), nasopharynx carcinoma (KB, ED50 = 1.85 micrograms/ml), colon carcinoma (COLO-205, ED50 = 2.24 micrograms/ml) and cervical carcinoma (HELA, ED50 = 1.92 micrograms/ml). Compounds 3 and 4 exhibited strong cytotoxicity against HEPA-3B, KB, COLO-205 and HELA (ED50 = 0.25, 1.81, 0.13 and 0.27 micrograms/ml for 3; ED50 = 0.87, 3.27, 0.56 and 0.35 micrograms/ml for 4, respectively.

Pharmacokinetics of honokiol after intravenous administration in rats assessed using high-performance liquid chromatography.

A simple and sensitive high-performance liquid chromatographic method for the identification and determination of honokiol in rat plasma has been developed. Up to 0.1 ml of plasma containing honokiol was deproteinized with acetonitrile, which contained an internal standard (paeonol). The supernatant was injected onto a reversed-phase C18 column using acetonitrile-water (70:30, v/v, adjusted to pH 2.5-2.8 with orthophosphoric acid) as the mobile phase and ultraviolet detection at 290 nm, followed by UV spectrum identification (between 220 and 380 nm) with a photodiode-array detector. The method was applied to pharmacokinetic studies of honokiol in rat following 5 or 10 mg/kg intravenous administration. A biphasic process consisting of a rapid distribution phase followed by a slower elimination phase was observed from the plasma concentration-time curves. Compartmental analysis yielded a two-compartment model.