OBJECTIVETo investigate the effects of nitrogen forms on the camptothecin (CPT) content, tryptophan synthase (TSB) and tryptophan decarboxylase (TDC) activities in Camptotheca acuminata seedlings.

METHODThe seedlings of C. acuminata with 6 pairs of leaves were subjected to 5 different NH4(+) -N/NO3(-) -N ratio (0 : 100, 75 : 25, 50 : 50, 25 : 75, 100 : 0) treatments by sand culture in a greenhouse. The CPT content, TSB activity in the young leaves and TDC in the stem barks of the seedlings were determined by HPLC on the 15th, 30th, 45th, 60th and 75th day, respectively.

RESULTThe obvious relationship between CPT content and nitrogen forms was observed. When NH4(+) - N /NO3(-) -N ratio was 25 : 75, CPT accumulation in young leaves displayed the best advantages (the highest value is 5.69 per thousand) and increased in the early 30 days of treatment and then declined. There was no obvious relationship between TSB activity in the young leaves and nitrogen forms. TDC activity in the stem bark was the highest when NH4(+) -N /NO3(-) -N ratio was 25 : 75, and the change of TDC activity paralleled to CPT content in the young leaves.

CONCLUSIONA short-term treatment that NH4(+) -N /NO3(-) -N ratio was 25:75 may gain high CPT content in the young leaves through enhancing the TDC activity in the stem bark of C. acuminata seedlings.

Aromatic-L-Amino-Acid Decarboxylases ; metabolism ; Camptotheca ; drug effects ; enzymology ; metabolism ; Camptothecin ; metabolism ; Drugs, Chinese Herbal ; metabolism ; Nitrogen ; chemistry ; pharmacology ; Plant Leaves ; drug effects ; enzymology ; metabolism ; Seedlings ; drug effects ; enzymology ; metabolism ; Tryptophan Synthase ; metabolism

OBJECTIVETo investigate the effects of nitrogen concentration on the camptothecin (CPT) content in Camptotheca acuminata seedlings:

METHODThe seedlings of C. acuminata with 6 pair of leaves were subjected to five nitrogen concentrations treatments by sand culture in a greenhouse. The CPT content in the seedlings was determined by HPLC on the 20th, 35th, 50th, 65th and 80th day respectively.

RESULTThe CPT content in the young leaves of C. acuminata seedlings supplied with different nitrogen concentration was significantly higher than that in other organs (P < 0.01), and it showed a single peak curve with the time course, the highest CPT content was observed on the 50th day after treatment. The CPT content in the young leaves obviously declined with increasing nitrogen concentration, and it reached the highest (6.72%) when nitrogen concentration was 4 mmol x L(-1), equal to 1.1 times that of 16 mmol x L(-1) nitrogen.

CONCLUSIONThe results demonstrate that proper deficient nitrogen stress can significantly enhance CPT accumulation in young leaves of C. acuminata seedlings.

Camptotheca ; drug effects ; metabolism ; Camptothecin ; metabolism ; Chromatography, High Pressure Liquid ; Nitrogen ; pharmacology ; Seedlings ; drug effects ; metabolism

Objective To investigate effects of atorvastatin on the development from macrophages (HMDM) to foam cells.Methods Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation and plastic adsorptive process.The isolated cells were stimulated by phorbol 12-myristate 13-acetate (PMA) (50 nmol/L) for 48 h and transformed to macrophages.Macrophages were co-incubated with 80 mg/L ox- idized low density lipoprotein (ox-LDL) and atorvastatin (0-100 ?mol/L),respectively for 0,6,12 and 24 h. Total cholesterol (TC),free cholesterol (FC) and protein (Pro) in cultured cells were quantitatively analyzed by high performance chromatography (HPLC) analysis and modified Lowry protein assay.Results When macropha- ges were incubated with 80 mg/L ox-LDL,the ratio of TC/Pro was greater than 20,and large amount of lipid drop- lets were displayed indicating the formation of foam cells.Atorvastatin decreased TC/Pro ratio in foam cells in a concentration and time dependent manner (0-100 ?mol/L)(P

OBJECTIVETo investigate the effect of hTERT antisense oligodeoxynucleotide (ASODN) on the oncogenicity and the inductive apoptosis of HL-60 cells.

METHODSApoptosis of HL-60 cells was detected by flow cytometry (FCM) and agarose gel electrophoresis. Both treated and untreated HL-60 cells were collected and transplanted into 5 BALB/c nude mice respectively, the formation of transplanted neoplasm and its morphologic change were observed. After the transplanted neoplasms were uniform with the ameliorated method in another 10 BALB/c nude mice, they were divided into 2 groups and injected ASODN and PBS into the neoplasm respectively. Seven days later, the tumor were measured, its morphology were observed, and the apoptotic cells were detected with a TUNEL kit.

RESULTSAfter 72 h treatment there were DNA ladders and early apoptosis peak in hTERT ASODN treated HL-60 cells but was none in SODN treated and blank control cells. In tumor formation experiment, neoplasms were formed in ASODN treated group at 16-17 d and untreated group at 12-13 d. Neoplasm was formed in 2 of 5 ASODN treated mice and 4 of 5 untreated mice respectively. In untreated mice tumor tissues were rich in blood vasa and stromal tissue compared with that in ASODN treated mice. In tumor therapy experiment, before treatment, there was no difference in the average neoplasm physical volume between ASODN treated group [(100.9 +/- 24.6) mm3] and PBS treated group [(98.4 +/- 23.1) mm3] (P > 0.05). After treatment, the neoplasm volume in ASODN treated group [(422.7 +/- 326.4) mm3] was smaller than that in PBS treated group [(786.4 +/- 357.6) mm3] (P < 0.05). Histologically, there were many apoptosis cells in ASODN treated group, but was seldom seen in PBS treated group. The TUNEL positive cells in ASODN treated group were much more than that in PBS treated group (P < 0.05).

CONCLUSIONThe hTERT ASODN induces apoptosis of HL-60 cells in vitro, reduces the tumor formation in BALB/c nude mice and inhibits the growth of the transplanted neoplasm.

Animals ; Apoptosis ; drug effects ; HL-60 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Telomerase ; genetics ; Transfection ; Xenograft Model Antitumor Assays

This study was purposed to investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN) on the proliferation and telomerase activity in HL-60 cells and to explore the relativity between the telomerase activity and the expression of hTERT gene in HL-60 cells. After treated by hTERT ASODN the expression of hTERT was detected by RT-PCR, the morphological changes of HL-60 cells was observed with inverted microscopy, the cell proliferation was measured by MTT method, and the telomerase activity was determined with TRAP-ELISA and TRAP-PAGE. The results showed that after sealing hTERT gene with ASODN for 72 hours, the expression of hTERT gene was significantly inhibited, the cell growth was repressed and the ability of proliferation decreased, and the effect was specific in sequence and dependent in dose and time. OD(450-690) values were 2.648 +/- 0.42, 1.504 +/- 0.47, 1.223 +/- 0.39, 0.944 +/- 0.16 respectively, as the cells were treated with 0, 10, 20, 30 micromol/L ASODN for 72 hours. The difference was significant as compared 10, 20, 30 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 20 micromol/L SODN group (2.376 +/- 0.65) with untreated group (2.648 +/- 0.42) (P > 0.05). TRAP-PAGE detection revealed that comparing ASODN groups with SODN groups the telomerase image bands were decreased and least was found in groups of 30 +/- mol/L. It is concluded that the hTERT ASODN may inhibit the proliferation and down-regulate the telomerase activity in HL-60 cells by sealing the expression of hTERT gene.
Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism ; pharmacology ; Transfection

Ten compounds were isolated from the barks of Jasminum giraldii by means of various of chromatographic techniques such as silica gel, Sephadex LH-20 and Rp-HPLC. Their structures were identified by spectroscopic data analysis as (+)-medioresinol (1), (+) -syringaresinol (2), syringaresinol-4'-O-beta-D-glucopyranoside (3), oleanic acid (4), 3-methoxy-4-hydroxy-trans-cinnamaldehyde (5), trans-sinapaldehyde (6), syringaldehyde (7), 1-(4-methoxy -phenyl) -ethanol (8), trans-cinnamic acid (9), and 4-(1-methoxyethyl) -phenol (10). Among them, compounds 1-3, 5-8 and 10 were isolated from the J. genus for the first time and compounds 4 and 9 were obtained from J. giraldii for the first time. In the DPPH free radical scavenging assay, compound 1 exhibited significant activity (IC50 55.1 micromol x L(-1)), compared with vitamin C(IC50 59.9 micromol x L(-1)); and compound 2 showed moderate activity (IC50 79.0 micromol x L(-1)), compared with 2, 6-di-tert-butyl4-methylphenol (IC50 236 micromol x L(-1)).
Antioxidants ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Jasminum ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVESTo analyze the differential expression genes (DEGs) among hepatocellular carcinoma (HCC), para-cancerous tissue (PCT) and normal liver tissue (NLT) and explore the target genes related to the development and progression of HCC.

METHODSThe total RNAs of matched HCC, PCT and NLT of HCC patients were isolated using one step Trizol method. Matched RNAs were qualified using 10 g/L agarose gel electrophoresis and lab-on-chip. cRNAs were synthesized, fluorescence labeled and purified after total RNAs were purified. The RNAs of HCC and NLT, HCC and PCT were hybridized with Agilent oligo microarray (21,074 probes). The fluorescence intensity features were detected by Agilent scanner and quantified by feature extraction software. The selected candidate genes were confirmed by SYBR Green I stained real time RT-PCR.

RESULTS(1) The total RNA, reverse transcription product and fluorescence labeled cRNA were all of high quality; (2) There were 420 up-regulated genes and 552 down-regulated genes among 2-fold DEGs, including DKK1 (dickkopf homolog 1) which was 5-fold up-regulated; (3) The results of real time RT-PCR, using beta-actin as an internal control, showed that the 2-Delta Ct values of DKK1 in HCC, PCT and NLT were 0.089 504, 0.007,65 and 0.000,631 respectively.

CONCLUSION(1) The high throughput and effective Agilent oligo microarray can screen novel therapy targeted genes by analyzing the DEGs in development and progression of HCC; (2) The development and progression of HCC is a complicated process involving multigenes and multiprocedures; (3) DKK1, as a novel gene, is involved in the development and progression of HCC and may be a new therapy target.

Carcinoma, Hepatocellular ; genetics ; pathology ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; pathology ; Oligonucleotide Array Sequence Analysis ; RNA, Neoplasm ; genetics

OBJECTIVETo study the effect of osteopontin (OPN) expression down-regulated by RNA interference (RNAi) on the invasiveness of hepatocelluar carcinoma cell line HCC-LM3.

METHODSHCC-LM3 cells were transfected with the chemically synthesized small interfering RNA (siRNA) formulated by lipofectamine 2000. Wild type HCC-LM3 and HCC-LM3 cells transfected with non-specific siRNA served as controls. Real-time PCR and Western blotting were used to quantify the mRNA and OPN protein levels. The malignant phenotypes of transfected HCC-LM3 cells including cellular growth rate, colony formation and Matrigel invasion activities were analyzed.

RESULTSSequence-specific siRNAs targeting OPN suppressed OPN RNA expression by 79% and also decreased OPN protein level by 81% in HCC-LM3 cells. The number of formed colonies and migrating numbers in vitro were decreased in HCC-LM3 cells transfected using sequence-specific siRNAs targeting OPN relative to controls (P < 0.05).

CONCLUSIONThis study demonstrated that specific siRNA is able to reduce OPN at both the mRNA and protein levels and significantly diminishes the invasiveness of hepatocellular carcinoma cells.

Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Down-Regulation ; Humans ; Liver Neoplasms ; genetics ; pathology ; Neoplasm Invasiveness ; Osteopontin ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo evaluate an enzymatic method for determining serum beta-hydroxybutyrate (beta-HB) with the National Committee for Clinical Laboratory Standards (NCCLS) projects, and to discuss its clinical values in diabetic ketoacidosis (DKA).

METHODSThe precision, accuracy, specificity, linearity and interference of the enzymatic method were analyzed. This method was used to determine serum beta-HB in 60 cases of normals, 50 cases of diabetes, and 34 cases of DKA by autochemistry analyzer.

RESULTSEnzymatic beta-HB assay was precise (within-run CV, day-to-day CV, and total CV < 5%). The linearity studies showed the method was linear up to 4 mmol/L. Recovery rate was 98.5%-104.1%. Hemolysis (Hemoglobin up to 18.2 g/L), icteric samples with total bilirubin up to 224 mumol/L, and lipemia up to triglyceride concentration of 2.28 mmol/L did not interfere with the beta-HB results in this method. Serum beta-HB levels were significantly elevated in DKA patients compared with DM patients and controls (P < 0.01). Positive rate of serum beta-HB in DKA patients was significantly higher than that of urinary ketone (P < 0.05).

CONCLUSIONSEnzymatic method is convenient and reliable, allows full automation, and is rapid enough to be used for both routine and urgent determinations of serum beta-HB. It can be used in diagnosing and monitoring treatment of DKA.

3-Hydroxybutyric Acid ; blood ; Adolescent ; Adult ; Autoanalysis ; Diabetes Mellitus ; blood ; Diabetic Ketoacidosis ; blood ; Evaluation Studies as Topic ; Female ; Humans ; Male ; Middle Aged

Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, Bartonella henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus-or species-bacteria were detected in 103 valid sequences. Eight species were edge type (Clone No. > 5). Three kinds of pathogens were identified (Borrelia burgdorferi, Bartonella henselae and Rickettsia sp). Three kinds of pathogens were not edge type(Clone No. < 5). Coverage value was 96.11%, and Shannon-Wiener index was 2.40. Analysis results of cloning sequence showed that tick-parasitic bacteria mainly were α and γ deformation mycetes which accounted for 56.25% (9/16). Conclusions The 16S rRNA gene sequences technology could make relative quantitative of bacterial flora, and detect many kinds of pathogens in tick. It's a good method for detection of pathogens and bacterial flora analysis.