Differentially proteomic analysis of the hemocytes against Aeromonas hydrophila infection in oriental river prawn Macrobrachium nipponense by iTRAQ approach.

As the direct executors of biological function, the expression level of proteins in host will reveal the molecular mechanisms regulating bacteria infection more directly. In the present study, the differential proteomes of Macrobrachium nipponense hemocytes response to Aeromonas hydrophila infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography electrospray ionization tandem mass spectrometry. The hemocyte proteins from the unchallenged and A. hydrophila challenged prawn, M. nipponense, at 12, 24 and 36 h post infection were compared. From this, a total of 3372 proteins were identified and 1014 proteins were considered differentially expressed, of which 117 common differentially expressed proteins were indicated between the time points. Hierarchical clustering, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment and protein-protein interaction network analyses were performed for the general characterization of overall enriched proteins. Cytoskeletal proteins including myosin heavy chain, myosin regulatory light chain, actin, tubulin alpha/beta chain, troponin I and troponin T as well as antioxidant enzymes such as catalase and cytosolic MnSOD were found significantly up-regulated in hemocytes, indicating that the phagocytosis process and ROS system were induced after challenge with A. hydrophila. And other proteins such as integrin ß, innexin inx2-like and heat shock protein 60 also participate in prawn immune response against bacteria. Parallel reaction monitoring analyses were carried out for validation of the expression levels of differentially expressed proteins, which indicated high reliability of the proteomic results. This is the first report on proteome of M. nipponense hemocytes against A. hydrophila infection, which contributes to better understanding on the molecular mechanisms of prawns.

A Study of the Relationship between the Polymorphism and Mutation of rs682429 and rs3781590 in the LRP5 Gene and Bone Metabolism in Postmenopausal Type 2 Diabetic Women in Xinjiang.

OBJECTIVE: To explore the expression of the polymorphism and mutation of rs682429 and rs3781590 in the low-density lipoprotein receptor-related protein 5 (LRP5) genotype and to analyse the relationship of bone mineral density (BMD) and bone metabolism markers in postmenopausal women with type-2 diabetes mellitus (T2DM) in Xinjiang, China, to provide a basis for prevention and treatment of the disease. METHODS: A total of 136 postmenopausal women were included in the study. According to the results of an oral glucose tolerance test (OGTT) and dual-energy X-ray (DEXA) determination of BMD, the study subjects were divided into 4 groups: group A: normal OGTT+normal bone mass group; group B: normal OGTT+osteoporotic (OP) group; group C: T2DM+normal bone mass group; group D: T2DM+osteoporotic (OP) group. Calcium (Ca), phosphorus (P), alkaline phosphatase (ALP), and clinical biochemical data were determined; haemoglobin A1c (HbA1c) was measured by HPLC; BMD of the femoral neck, hip, and lumbar spine (L1-4) was measured by dual-energy X-ray (DEXA); and the rs682429 and rs3781590 polymorphisms of the LRP5 gene were detected by time-of-flight mass spectrometry (TOF MS). RESULTS: (1) The rs682429 polymorphism of the LRP5 genotype distribution was statistically significant (P < 0.05) in group B compared with group A. (2) The triglycerides (TG) of women with the CT/TT genotype (mutant type) were higher than those of women with the CC genotype (wild type) (2.37 ± 1.30 vs. 1.52 ± 0.83, P < 0.05) at the rs3781590 site of the LRP5 gene in group D. (3) Multiple linear regression analysis showed that TG (ß = 0.034, P < 0.05) and body mass index (BMI) (ß = 0.013, P < 0.05) were the influencing factors of BMD (L1-4) in T2DM patients. TG (ß = 0.022, P < 0.05), BMI (ß = 0.009, P < 0.05), and duration of menopause (ß = 0.005, P < 0.05) were the influencing factors of BMD (hip). CONCLUSION: (1) The rs682429 polymorphism site in the LRP5 gene may be involved in bone metabolism in postmenopausal women from Xinjiang. (2) The rs3781590 mutation in the LRP5 gene from these subjects may be involved in lipid metabolism. (3) Among postmenopausal women with type 2 diabetes mellitus and bone mass abnormality in the Xinjiang Shihezi area, high BMI and TG are protective factors against increased BMD. Duration of menopause is a risk factor for increased BMD.

A meta-analysis of single-case research on the use of tablet-mediated interventions for persons with ASD.

BACKGROUND: There is a growing amount of single-case research literature on the benefits of tablet-mediated interventions for individuals with autism spectrum disorder (ASD). With the development of tablet-based computers, tablet-mediated interventions have been widely utilized for education and treatment purposes; however, the overall quality and evidence of this literature-base are unknown. AIMS: This article aims to present a quality review of the single-case experimental literature and aggregate results across studies involving the use of tablet-mediated interventions for individuals with ASD. METHODS AND PROCEDURES: Using the Tau nonoverlap effect size measure, the authors extracted data from single-case experimental studies and calculated effect sizes differentiated by moderator variables. The moderator variables included the ages of participants, participants' diagnoses, interventions, outcome measures, settings, and contexts. OUTCOMES AND RESULTS: Results indicate that tablet-mediated interventions for individuals with ASD have moderate to large effect sizes across the variables evaluated. The majority of research in this review used tablets for video modeling and augmentative and alternative communication. CONCLUSIONS AND IMPLICATIONS: To promote the usability of tablet-mediated interventions for individuals with ASD, this review indicates that more single-case experimental studies should be conducted with this population in naturalistic home, community, and employment settings.

NFATC1 promotes cell growth and tumorigenesis in ovarian cancer up-regulating c-Myc through ERK1/2/p38 MAPK signal pathway.

It has been reported that nuclear factor of activated T cells (NFATC1) was up-regulated in cancers mediating malignant behaviors. However, the role of NFATC1 in ovarian cancer has not been elucidated. In the present study, we undertook to explore the clinicopathological significance of NFATC1 expression and the mechanism by which NFATC1 works in ovarian cancer. Expression status of NFATC1 was examined using immunohistochemistry. Both knockdown and re-expression of NFATC1 on ovarian cancer cells were employed to observe the effect overgrowth. It was found that NFATC1 was significantly overexpressed in ovarian cancer tissues in comparison with paired normal control tissues and that overexpression of NFATC1 was significantly associated with metastasis and poor prognosis on clinical tissue level. In in vitro ovarian cancer cell lines, we found that NFATC1 can promote proliferation up-regulating c-myc through activation of ERK1/2/p38/MAPK signal pathway. Together, the results we obtained demonstrated that NFATC1 played oncogenic role in ovarian cancer. Mechanistically, NFATC1 promoted growth of ovarian cancer cells up-regulating c-myc through activation of ERK1/2/p38/MAPK signal pathway, suggesting that NFATC1 might be used as a therapeutic target for ovarian cancer.

Deacetyl-mycoepoxydiene, isolated from plant endophytic fungi Phomosis sp. demonstrates anti-microtubule activity in MCF-7 cells.

Deacetyl-mycoepoxydiene (DM), a novel secondary metabolite produced by the plant endophytic fungi Phomosis sp., induced the reorganization of cytoskeleton in actively growing MCF-7 cells by promoting polymerization of tubulin. DM could induce cell cycle arrest at G2/M in MCF-7 cells. Additionally, DM-induced apoptosis was characterized with up-regulating caspase-3, Bax, caspase-9, parp, and p21 while down-regulating Bcl-2 activation. DM conferred dose- and time-dependent inhibitory effects upon cell proliferation of MCF-7 cells both in cultured cells and nude mice with human breast carcinoma xenografts. The results obtained from these in vitro and in vivo models provide new data revealing the potential for DM as a novel microtubule inhibitor.

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, inhibits human oral squamous carcinoma cells KB and KB/VCR: In vitro and in vivo studies.

BACKGROUND: Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, might possess anti-cancer properties. We aimed to evaluate the efficacy of Riccardin D-26 as a candidate compound for treatment of cancers with sensitive or drug resistant cells. METHODS: Experiments were performed on human oral squamous carcinoma KB cells and vincristin-selected MDR KB/VCR cells. The inhibition of cell growth was evaluated by colorimetric and clonogenic assays. The apoptotic cells were determined by the Annexin V-FITC/PI staining assay. JC-1 fluorescence probe was used to examine the mitochondria membrane potential (MMP). Further experiments were performed in nude mice bearing KB or KB/VCR xenografts. Riccardin D-26 was administered by injection for 2weeks. The specimens of KB and KB/VCR xenografts were removed for TUNEL staining and Western blotting analysis. RESULTS: Riccardin D-26 significantly inhibited cancer growth in both KB and KB/VCR cells. Riccardin D-26's activity in cancer cells was greater than that in human normal liver cells. In mice, Riccardin D-26 effectively prevented the growth of KB and KB/VCR xenografts without significant toxicity. Further studies suggested that Riccardin D-26 inhibited cancer growth by inducing apoptosis in the activation of mitochondria-mediated intrinsic apoptosis pathway. Riccardin D-26 also possessed this activity in regulation of mitogen-related protein kinases such as MAPK and PI3K/Akt, which is associated with its inhibitory effect on KB/VCR cells. CONCLUSIONS: Riccardin D-26 possessed an anti-proliferation activity against both sensitive KB and MDR KB/VCR cancer cells. GENERAL SIGNIFICANCE: Riccardin D-26 could be a promising agent for treatment of cancers with sensitive or drug resistant cells.

Microbial biotransformation of kurarinone by Cunninghamella echinulata AS 3.3400.

In this paper, microbial transformation of kurarinone (1) by Cunninghamella echinulata AS 3.3400 was investigated and four transformed products were isolated and identified as 6″-hydroxykurarinone (2), 4″,5″,8″-trihydroxynorkurarinone (3), norkurarinone (4), and kurarinone-7-O-ß-glucoside (5), respectively. Among them, 3 and 5 are new compounds, and the rare glycosylation in microbial transformation was observed. In addition, the cytotoxicities of transformed products (2-5) were also investigated.

Microbial transformation of Norkurarinone by Cunninghamella blakesleana AS 3.970.

In this paper, microbial transformation of norkurarinone (1) by Cunninghamella blakesleana AS 3.970 was investigated and seven transformed products were isolated and characterized as kurarinone (2), 4″,5″-dihydroxykurarinone (3), 6″-hydroxyl-2'-methoxyl-norkurarinone 7-O-ß-d-glucoside (4), 6″-hydroxyl-norkurarinone 4'-O-ß-d-glucoside (5), 4″,5″-dihydroxynorkurarinone (6), 7-methoxyl-norkurarinone (7), and 7-methoxyl-4″,5″-dihydroxynorkurarinone (8), respectively. Among them, 3-5 are new compounds, and the glycosylation reaction in microbial transformation process was reported rarely. In addition, the cytotoxicities of transformed products (1-8) were also investigated.

Improved lycopene production by Blakeslea trispora with isopentenyl compounds and metabolic precursors.

The highest lycopene production in mated cultures of Blakeslea trispora was 578 mg/l by adding 42 mg geraniol/l to the medium after 48 h of growth. The control gave 317 mg/l. Adding isopentenyl alcohol at 40 mg/l, mevalonic acid at 17.5 mg/l or dimethyl allyl alcohol at 150 mg, each after 36 h growth, gave lycopene yields 62, 45 and 47%, respectively, higher than the control.

Overexpression of chromokinesin KIF4 inhibits proliferation of human gastric carcinoma cells both in vitro and in vivo.

Gastric carcinoma is a common type of malignant tumors and is associated with high death rates. The pathogenesis of gastric carcinoma is still unclear, and increasing evidence shows that many factors contribute to this process. Chromokinesin KIF4 is involved in multiple critical cellular processes. Recently, it has become apparent that KIF4 plays a crucial suppressive role in tumorigenesis. However, the role of KIF4 in human gastric cancer is still unclear. In this study, we examined expression profiles of KIF4 in gastric carcinoma specimens and generated gastric cancer cells that stably express GFP-KIF4 fusion protein (designated as BGC-GFP-KIF4 cells) followed by cell proliferation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and soft agar colony-formation assays. Simultaneously, we further examined the capability of tumor formation of BGC-GFP-KIF4 cells in nude mice. The results showed that among 23 gastric carcinoma specimens, 13 cases (56.6%) had lower expression of KIF4 compared with corresponding adjacent tissues. In addition, there was a significant correlation between low expression of KIF4 and poor differentiation of tumor (P = 0.024). Overexpression of KIF4 in BGC cells inhibited cell proliferation in vitro, as well as their ability to form tumors in vivo. Our findings suggest that human chromokinesin KIF4 functions as an inhibitor of gastric cancer cell proliferation and might serve as a novel biological target to cure human gastric carcinoma.

Effect of chlorogenic acid on mast cell-dependent anaphylactic reaction.

Chlorogenic acid (CGA), a naturally occurring polyphenol compound, has a number of biological activities. However, roles of CGA in the mast cell-dependent anaphylactic reaction have not been fully examined. In the present study, the effect and mechanism of CGA on mast cell-dependent anaphylactic reaction were investigated using in vivo and in vitro models. CGA inhibited compound 48/80-induced systemic anaphylactic shock in mice and skin vascular permeability in rats. CGA also inhibited anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis (PCA). Moreover, CGA dose-dependently reduced histamine and TNF-alpha release from RBL-2H3 cells activated by anti-DNP IgE. Pretreatment with CGA suppressed IgE-antigen complex induced calcium uptake into RBL-2H3 cells. When CGA was added, the level of intracellular cyclic adenosine monophosphate (cAMP) in RBL-2H3 cells was significantly elevated compared with the untreated cells. Decreased calcium uptake and increased cAMP level might be involved in the inhibitory effect of CGA on mast cell activation. These results suggest a possible therapeutic application of CGA in allergic diseases.

Modulation of P-glycoprotein activity by the substituted quinoxalinone compound QA3 in adriamycin-resistant K562/A02 cells.

QA3 is a derivative of the substituted 1,3-dimethyl-1H-quinoxalin-2-ones, which are compounds that may selectively antagonize P-glycoprotein (P-gp) in multidrug resistance (MDR) cancer cells. Our previous work identified QA3 as a candidate compound for reversing MDR in cancer cells. In the present study, we found that QA3 significantly decreases the intracellular level of ATP, stimulates ATPase activity in membrane microsomes and decreases protein kinase C (PKC) activity. These results indicated that QA3 inhibits P-gp activity by blocking ATP hydrolysis and ATP regeneration. Furthermore, QA3 triggered and increased adriamycin-induced K562/A02 cell apoptosis as evidenced by Annexin V-FITC plus PI staining.Western blot analysis showed that the levels of cleaved caspase-9 and cleaved caspase-3 proteins increased, and similarly, the levels of procaspase-9 and procaspase-3 decreased after QA3 treatment. Consequently, poly ADP-ribose polymerase (PARP) activity increased as evidenced by the presence of the PARP cleavage product in K562/A02 cells. QA3 also enhanced the potency of adriamycin against K562/A02 cells as demonstrated by increased apoptosis and activation of caspase-9,-3 and PARP. These data support the observation that P-gp activity is inhibited after QA3 treatment. Moreover, these results indicate that QA3 is a novel MDR reversal agent with potent inhibitory action against P-gp MDR cancer cells.

In vitro and in vivo evaluation of silybin nanosuspensions for oral and intravenous delivery.

In this study, we evaluate the effect of particle sizes on the physicochemical properties of silybin and identify the influence of silybin nanosuspensions on its permeation across the Caco-2 cell monolayer. In vivo pharmacokinetic evaluation of silybin nanosuspensions was also carried out in beagle dogs. TEM, AFM and SEM analyses revealed the effect of homogenization pressure on particle size and morphology, and confirmed the existence of a surfactant-stabilizer film on the surface of nanoparticles. DSC and XRPD experiments manifested that the crystalline state was maintained as particle size was reduced and the enhanced dissolution property was due to the increased surface area. Nanosuspensions had a significant influence on drug transport across the Caco-2 cell monolayer and the enhanced dissolution velocity was responsible for the increased permeability. A pharmacokinetics study in beagle dogs further confirmed the in vitro results and demonstrated that oral administration of silybin nanosuspensions significantly increase its bioavailability compared to the coarse powder. Nanosuspensions of silybin with smaller particle size reveal a higher potential to increase their oral bioavailability; while for intravenous infusion the lower pressure produced silybin nanosuspensions appeared to maintain a more sustained drug release profile.

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice.

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA-PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.

Marchantin C: a potential anti-invasion agent in glioma cells.

Cancer cell migration is a leading cause of tumor recurrence and treatment failure. Previously, we reported that marchantin C exhibited promising antitumor activity by inducing microtubule depolymerization and apoptosis. In the present study, we investigated the effect of marchantin C on inhibition of migration in T98G and U87 cells. The scratch-induced migration, Boyden chamber and cell invasion assays were applied to determine that the migrating capacity and invasiveness of these glioma cell lines were inhibited when exposed to marchantin C at a low concentration. There are no obvious signs of apoptosis with this dose. Western blot analyses confirmed that MMP-2, a key role in cancer cell migration, was reduced after incubation with marchantin C in both glioma cell lines. In addition, signaling pathway investigations demonstrated that ERK/MAPK might be involved in MMP-2 downregulation, rather than the AKT/PI3K or JAK/STAT3 pathways. Moreover, marchantin C potently suppressed angiogenesis activity in vivo by CAM assay. This is the first study to demonstrate that marchantin C can inhibit glioma cell migration and invasiveness.

Marchantin C, a novel microtubule inhibitor from liverwort with anti-tumor activity both in vivo and in vitro.

Microtubules are long-standing targets in cancer chemotherapy. Previously, we reported that marchantin C triggers apoptosis of human tumor cells. We show here that marchantin C induced cell cycle arrest at G(2)/M phase in A172 and HeLa cells. In addition, marchantin C decreased the quantity of microtubules in a time- and dose-dependent manner in these cells. Exposure of purified bovine brain tubulin to marchantin C inhibited polymerization of gross tubulin in vitro. Moreover, marchantin C potently suppressed the growth of human cervical carcinoma xenografts in nude mice. Marchantin C-treated xenografts showed decreased microtubules, Bcl-2 and increased cyclin B1, Bax, caspase-3, indicating that marchantin C possess the same ability to induce microtubules depolymerization and tumor cell apoptosis in tumor-bearing mice as in vitro. In conclusion, marchantin C is a novel microtubule inhibitor that induces mitotic arrest of tumor cells and suppresses tumor cell growth, exhibiting promising antitumor therapeutic potential.

Reversal of p-glycoprotein-mediated multidrug resistance by macrocyclic bisbibenzyl derivatives in adriamycin-resistant human myelogenous leukemia (K562/A02) cells.

Macrocyclic bisbibenzyls, a class of characteristic natural molecules derived from liverworts, have diverse biological significances. Dihydroptychantol A (DHA) was identified to be an antifungal active macrocyclic bisbibenzyl from liverwort Asterella angusta. In an attempt to understand other biological activities of this compound, the chemical synthesized DHA and its analogues (compounds 1-3) were employed to test this possibility by using adriamycin-resistant K562/A02 cells. Among the tested compounds (1-4), DHA showed the strongest potency to increase adriamycin cytotoxicity toward K562/A02 cells by MTT assays and its reversal fold is 8.18 (20 microM). Mechanisms of DHA on p-glycoprotein (P-gp)-mediated multidrug resistance (MDR) were further investigated. Based on the flow cytometry, we detected the significant increase of adriamycin and rhodamine123 accumulation in K562/A02 cells exposed to various concentrations of DHA, meanwhile, notable decrease of rhodamine123 efflux was also observed, which revealed DHA caused a decline of P-gp activity. Furthermore, P-gp expression was analyzed by the flow cytometry and RT-PCR. Dose-dependent reduction of P-gp expression was measured in K562/A02 cells pretreated with DHA for 24h. No such results were found in parental K562 cells. These results demonstrated DHA reversed effectively MDR by blocking the drugs to be pumped out via inhibiting P-gp function and expression pathway.

Reversal effect of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in adriamycin-resistant K562/A02 cells.

Plagiochin E is a new macrocyclic bisbibenzyl compound isolated from Marchantia polymorpha. In the previous studies, we reported that when combined with fluconazole, plagiochin E had synergetic effects against the resistant strain of Candida albicans. Herein, we examined the reversal effect of plagiochin E on multidrug resistance in adriamycin-induced resistant K562/A02 cells and the parental K562 cells. Its cytotoxicity and reversal effects on multidrug resistance were assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assay. Apoptosis percentage of cells was obtained from Annexin V/fluorescein isothiocyanate (FITC) and propridium iodide (PI) double-staining. The effects of plagiochin E on P-glycoprotein activity were evaluated by measuring rhodamine 123 (Rh123)-associated mean fluorescence intensity and P-glycoprotein expression on the basis of the flow cytometric technology, respectively. The results showed that plagiochin E ranging from 2 to 12 mug/ml had little cytotoxicity against K562/A02 cells. When combined with adriamycin, it significantly promoted the sensitivity of K562/A02 cells toward adriamycin through increasing intracellular accumulation of adriamycin in a dose-dependent manner. Further study demonstrated that the inhibitory effect of plagiochin E on P-glycoprotein activity was the major cause of increased stagnation of adriamycin inside K562/A02 cells, indicating that plagiochin E, as a new class of mutidrug resistance inhibitor, may effectively reverse the multidrug resistance in K562/A02 cells via inhibiting expression and drug-transport function of P-glycoprotein.

Marchantin C, a macrocyclic bisbibenzyl, induces apoptosis of human glioma A172 cells.

Macrocyclic bisbibenzyls, a class of characteristic components derived from liverworts, are attracting more and more attention because of their wide range of biological significance, including anti-bacterial, anti-fungus, anti-oxidation and cytotoxicity. Herein, we investigated the pro-apoptotic effect of marchantin C on human glioma A 172 cells. The results demonstrated that marchantin C conferred dose-dependent inhibitory effects onto cell growth, viability and colony formation ability of A 172 cells. Morphological observation and DNA laddering assay showed that marchantin C-treated A172 cells displayed outstanding apoptosis characteristics, such as nuclear fragmentation, the appearance of membrane-enclosed apoptotic bodies and DNA laddering fragment. Moreover, flow cytometric detection of phosphatidylserine externalization indicated that marchantin C-induced apoptosis occurred in a dose-dependent manner. RT-PCR and western blot assay further substantiated that marchantin C, as a promising pro-apoptotic agent, had strong effects to induce A172 cell apoptosis, suggesting that the action was achieved through up-regulating Bax and down-regulating Bcl-2.