OBJECTIVETo extract RNA from Pinellia ternata and lay a foundation for studying the formation mechanism of P. ternata.

METHODBy modifying the method recommended by Guanidinium for extracting total RNA from plant tissues rich in phenolic and polysaccharidic compounds, a simple and convenient method for extraction of total RNA from the tubers, stems and leaves of P. ternate containing abundant polyphenols and polysaccharides was established. High concentrated p-mercaptoethanol was added in the RNA extracted buffer to remove polyphenols, phenol and chloroform were used to eliminate proteins, and isopropanol and sodium acetate were used to precipitate polysaccharides.

RESULTThe A260/A230 value of RNA extracted with improved method were all over 2.0 and the values of A260/A280 were between 1.7 and 2.0. The electrophoresis bands were cleared on agarosegel and integrity of RNA was good.

CONCLUSIONThe results showed that RNA obtained from the tubers, stems and leaves of P. ternate with this method had high purity and quality and could be used in molecular biological research, as DDRT-PCR and reverse Northern blotting analysis directly. This method is simple, economic, stable performance, and has a good repeatability as well as is suitable for extracting total RNA of medicinal plants with high concentrations of phenolics and polysaccharides.

Blotting, Northern ; Pinellia ; genetics ; Plant Leaves ; genetics ; Plant Stems ; genetics ; Plant Tubers ; genetics ; Polymerase Chain Reaction ; RNA, Plant ; isolation & purification

Pathological cardiac hypertrophy is a maladaptive response in a variety of organic heart disease(OHD),which is characterized by mitochondrial dysfunction that results from disturbed energy metabolism. SIRT3, a mitochondria-localized sirtuin, regulates global mitochondrial lysine acetylation and preserves mitochondrial function. However, the mechanisms by which SIRT3 regulates cardiac hypertrophy remains to be further elucidated. In this study, we firstly demonstrated that expression of SIRT3 was decreased in AngiotensionⅡ(AngⅡ)-treated cardiomyocytes and in hearts of AngⅡ-induced cardiac hypertrophic mice. In addition, SIRT3 overexpression protected myocytes from hypertrophy, whereas SIRT3 silencing exacerbated Ang II-induced cardiomyocyte hypertrophy.In particular,SIRT3-KO mice exhibited significant cardiac hypertrophy. Mechanistically, we identified NMNAT3 (nicotinamide mononucleotide adenylyltransferase 3), the rate-limiting enzyme for mitochondrial NAD biosynthesis, as a new target and binding partner of SIRT3.Specifically,SIRT3 physically interacts with and deacety-lates NMNAT3,thereby enhancing the enzyme activity of NMNAT3 and contributing to SIRT3-mediated anti-hypertrophic effects.Moreover,NMNAT3 regulates the activity of SIRT3 via synthesis of mitochon-dria NAD.Taken together,these findings provide mechanistic insights into the negative regulatory role of SIRT3 in cardiac hypertrophy.Sirtuin 3(SIRT3),a mitochondrial deacetylase that may play an impor-tant role in regulating cardiac function and a potential target for CHF

Objective: To investigate the relationship between P27，P53 and PCNA expression in human gastric carcinoma tissues and clinicopathological parameters. Methods: The expression of P27，P53 and PCNA in 62 human gastric carcinoma tissues was examined with immunohistochemistry SP method. Results: Positive rates of P27，P53 and PCNA expression were 37.1％, 40.4%，83.9％. P27 expression was related with Bormann type, infiltrative depth, lymph node and distant metastasis and clinical stage. P53 expression was related with sex of patients, distant metastasis and clinical stage. PCNA expression was related with age of patients and infiltrative depth of tumor. P27 positive expression group was higher than negative group as to 5-year survival. P27 expression was in reverse relation with PCNA expression. Conclusion: The expression of P27, P53 and PCNA may be regarded as an important marker in judging malignant degree of gastric carcinoma,distant metastasis and prognosis.

BACKGROUNDIt has been reported that CD8(+) regulatory cells could be induced upon oral tolerance. The purpose of this study was to investigate the changes of CD8α(+) T cells in dextran sulfate sodium (DSS)-induced colitis mice pretreated by oral immune regulation.

METHODSThe effects of five low oral doses of colitis-extracted proteins (CEP) on colitis were evaluated by clinical manifestation and histological lesions. The percentages of CD8α(+) T cells gating on CD3(+) T cells were evaluated in the gut-associated lymphoid tissues (GALT) and the spleens by flow cytometry. Differences between the two groups were compared by Student's t test or Mann-Whitney U test.

RESULTSCompared to bovine serum albumin (BSA)-fed control mice, administration of CEP resulted in marked alleviation of colitis. The proportion of CD8α(+) T cells, not only in intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of the large intestine (LI) but also in spleen from CEP-fed colitis mice, was significantly higher than that from BSA-fed colitis mice (LI-IELs: (71.5 ± 5.4)% vs. (60.1 ± 4.3)%, P < 0.01; LI-LPLs: (60.7 ± 5.2)% vs. (51.9 ± 4.7)%, P < 0.01; spleen: (24.1 ± 3.6)% vs. (20.3 ± 4.1)%, P < 0.05; n = 8). Mucosal repair in repair-period mice five days after termination of DSS treatment was also accompanied by an increase of CD8α(+) T cells in large intestinal mucosal lymphocytes (LI-IELs: (72.1 ± 3.7)% vs. (61.5 ± 4.5)%, P < 0.01; LI-LPLs: (62.1 ± 5.7)% vs. (52.7 ± 3.6)%, P < 0.01; n = 8). The proportion of CD3(+) T cells increased in Peyer's patches (PPs) and decreased in mesenteric lymph nodes (MLNs) from colitis mice compared to untreated mice, whereas the change pattern of CD3(+) T cells in PPs and MLNs from CEP-fed colitis mice was just on the contrary.

CONCLUSIONImprovement of DSS-induced colitis resulted from oral immune regulation is associated with an increase in CD8α(+) T cells in spleen and large intestinal mucosa.

Administration, Oral ; Animals ; CD8-Positive T-Lymphocytes ; metabolism ; Colitis ; chemically induced ; complications ; Dextran Sulfate ; toxicity ; Flow Cytometry ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Proteins ; administration & dosage ; immunology ; Spleen ; cytology ; metabolism

OBJECTIVETo review the current advances on the role of uncoupling protein (UCP) in the pathogenesis and progress of nonalcoholic fatty liver disease (NAFLD).

DATA SOURCESA comprehensive search of the PubMed literature without restriction on the publication date was carried out using keywords such as UCP and NAFLD.

STUDY SELECTIONArticles containing information related to NAFLD and UCP were selected and carefully analyzed.

RESULTSThe typical concepts, up-to-date findings, and existing controversies of UCP2 in NAFLD were summarized. Besides, the effect of a novel subtype of UCP (hepatocellular down regulated mitochondrial carrier protein, HDMCP) in NAFLD was also analyzed. Finally, the concept that any mitochondrial inner membrane carrier protein may have, more or less, the uncoupling ability was reinforced.

CONCLUSIONSConsidering the importance of NAFLD in clinics and UCP in energy metabolism, we believe that this review may raise research enthusiasm on the effect of UCP in NAFLD and provide a novel mechanism and therapeutic target for NAFLD.

Animals ; Fatty Acids, Nonesterified ; metabolism ; Fatty Liver ; etiology ; metabolism ; Humans ; Ion Channels ; physiology ; Mitochondrial Proteins ; analysis ; chemistry ; physiology ; Non-alcoholic Fatty Liver Disease ; Uncoupling Protein 2

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.
Bacillus subtilis ; enzymology ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Activation ; drug effects ; Hydrogen-Ion Concentration ; Kinetics ; Mannosidases ; chemistry ; isolation & purification ; metabolism ; Mercury ; pharmacology

OBJECTIVETo investigate whether apollon immunoexpression in breast cancer tissues helps to predict pathological complete response (pCR) after neoadjuvant chemotherapy (NAC).

METHODSThe expressions of Apollon, Her-2, estrogen receptor (ER) and progesterone receptor (PR) were detected immunohistochemically in biopsy tissues from 124 breast cancer patients. The clinical responses to NAC were evaluated in line with the response evaluation criteria in solid tumors (RECIST). The pCR rate was analyzed for different types of breast cancer. The correlations between Apollon status with Her-2, ER, PR, lymph node status and tumor size were analyzed. Immunohistochemistry was used to compared the changes in Apollon expression in the breast cancer tissues before and after NAC.

RESULTSThe pCR rate was 18.5% (23/124) in these patients. Negative expressions of apollon, ER and PR were all associated with a higher pCR rate after NAC. Apollon was significantly correlated with Her-2, ER, PR and lymph node involvement. Chemotherapy significantly down-regulated apollon expression in the tumor cells.

CONCLUSIONA negative apollon expression might be a predictor of pCR in patients with breast cancer.

Biopsy ; Breast Neoplasms ; drug therapy ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Inhibitor of Apoptosis Proteins ; metabolism ; Neoadjuvant Therapy ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

To decrease the hemolysis side effect of puerarin, the basic formula and preparation of puerarin submicron emulsion were optimized and the physicochemical properties were evaluated. Puerarin submicron emulsions were prepared by phase inversion-ultrasound combining with phospholipids complexes technology. The effects of preparative parameters, such as emulsification time, stirring velocity and ultrasound time, on mean diameter, span of dispersity, entrapment efficiency and overall desirability were investigated. The three dimensional response surface graphs were produced by second-order polynomial and liner equation, which predict the optimal experiment conditions. All response variables were found to be greatly dependent on three independent variables. Second-order polynomial equations were fitter than liner equations for this study. The optimal emulsification time, stirring velocity and ultrasound time was 15 min, 2 000 r x min(-1), 30 min, respectively. The mean diameter, span of dispersity, entrapment efficiency, drug content and zeta potential of emulsions prepared by the method were 228.23 nm, 0.628 4, 84. 32%, 9.98 mg x mL(-1), - 29.03 mV, respectively. Puerarin submicron emulsion was prepared by the optimized preparation method. The narrow particle diameter distribution, high envelopment efficacy and good stability were obtained. The physicochemical properties were suitable for the requirement of the intravenous emulsion.
Emulsions ; Isoflavones ; administration & dosage ; chemistry ; Particle Size

Animals ; Flavonoids ; pharmacology ; Interleukin-10 ; biosynthesis ; genetics ; Liver ; metabolism ; Liver Diseases, Alcoholic ; metabolism ; Phenols ; pharmacology ; Polyphenols ; Rats ; Rats, Sprague-Dawley ; Tea ; chemistry

OBJECTIVETo explore the mechanism of Alpha-fetoprotein (AFP) effects on hepatocellular carcinoma cells (HCC) resistances apoptosis induced by tumor necrosis factor-related apoptosis inducing-ligand (TRAIL).

METHODSThe expressed alteration of TRAIL receptor-2 (DR5) after the human hepatoma cells line Bel 7402 (AFP-producing) and HLE cells (non-AFP producing) were treated with all trans retinoic acid (ATRA) were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation (Co-IP); Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering (RNAi) was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP (named pcDNA3.1-afp); The growth of hepatoma cells was analyzed by MTT.

RESULTSBel 7402 and HLE cells expressed DR5, lowed dosage of ATRA (40mumol/L) had no influence on the expression of DR5 in Bel 7402 cells, but ATRA (160mumol/L) could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with RAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced.

CONCLUSIONSThese data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistance-induced apoptosis by TRAIL.

Apoptosis ; Cell Line, Tumor ; metabolism ; Humans ; Receptors, Retinoic Acid ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Tretinoin ; pharmacology ; alpha-Fetoproteins ; metabolism