OBJECTIVETo study the effects of matrine on gamma-globin synthesis in K562 cells in vitro.

METHODSBenzidine staining was used to study the dose- and time-dependent effects of matrine on hemoglobin synthesis in K562 cells, and Western blotting was performed to determine the level of hemoglobin F(alpha(2)gamma(2)).

RESULTSBenzidine staining showed that K562 cells treated with matrine of 0.1 mg/ml had a positivity rate for benzidine (BZ%) of 15.67% at 96 h after the treatment, and Western blotting indicated increased synthesis of hemoglobin F.

CONCLUSIONMatrine can induce gamma-globin synthesis and increase hemoglobin F level in K562 cells, the effect of which resembles that of sodium butyrate.

Alkaloids ; pharmacology ; Blotting, Western ; Fetal Hemoglobin ; biosynthesis ; Globins ; biosynthesis ; Humans ; K562 Cells ; Quinolizines ; pharmacology

Fungal elicitors are a group of chemicals that can stimulate the secondary metabolite production in plants and microbial cells. After being recognized, it could enhance the expression of related genes through the signal-transduction pathway; regulate the activity of the enzyme involved in the biosynthesis of secondary metabolites. In recent years, the inducible mechanism of fungal elicitors has been studied deeply worldwide. Meanwhile, it has acquired wide concern in the area of biological industry, especially in the fermentation industry. This paper addresses the application and prospect of fungal elicitors in the secondary metabolites of plant and microbial cells.
Alkaloids ; biosynthesis ; genetics ; Camptothecin ; biosynthesis ; Fermentation ; Fungi ; genetics ; metabolism ; Industrial Microbiology ; methods ; Paclitaxel ; biosynthesis ; Plants, Medicinal ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Secondary Metabolism

Transcription factor is one of the key factors in the regulation of gene expression at the transcriptional level. It plays an important role in plant growth, active components biosynthesis and response to environmental change. This paper summarized the structure and classification of bHLH transcription factors and elaborated the research progress of bHLH transcription factors which regulate the active components in plants, such as flavonoids, alkaloids, and terpenoids. In addition, the possibility of increasing the concentration of active substances by bHLH in medicinal plants was assessed. The paper emphasized great significance of model plants and multidisciplinary research fields including modern genomics, transcriptomics, metabolomics and bioinformatics, providing the contribution to improve the discovery and function characterization of bHLH transcription factors. Accelerating the research in the mechanism of bHLH transcription factors on the regulation of active components biosynthesis will promote the development of breeding and variety improvement of Chinese medicinal materials, also ease the pressure of resources exhaustion of traditional Chinese medicine home and abroad.
Alkaloids ; biosynthesis ; Basic Helix-Loop-Helix Transcription Factors ; chemistry ; classification ; genetics ; metabolism ; Flavonoids ; biosynthesis ; Plants, Medicinal ; genetics ; metabolism ; Terpenes ; metabolism

Terpenoid indole (TIAs) and β-carboline alkaloids (BCAs), such as suppressant reserpine, vasodilatory yohimbine, and antimalarial quinine, are natural compounds derived from strictosidine. These compounds can exert powerful pharmacological effects but be obtained from limited source in nature. the whole biosynthetic pathway of TIAs and BCAs, The Pictet-Spengler reaction catalyzed by strictosidine synthase (STR; EC: 4.3.3.2) is the rate-limiting step. Therefore, it is necessary to investigate their biosynthesis pathways, especially the role of STR, and related findings will support the biosynthetic generation of natural and unnatural compounds. This review summarizes the latest studies concerning the function of STR in TIA and BCA biosynthesis, and illustrates the compounds derived from strictosidine. The substrate specificity of STR based on its structure is also summarized. Proteins that contain six-bladed four-stranded β-propeller folds in many organisms, other than plants, are listed. The presence of these folds may lead to similar functions among organisms. The expression of STR gene can greatly influence the production of many compounds. STR is mainly applied to product various valuable drugs in plant cell suspension culture and biosynthesis in other carriers.
Alkaloids/biosynthesis* ; Carbolines/metabolism* ; Carbon-Nitrogen Lyases ; Indoles/metabolism* ; Terpenes/metabolism*

OBJECTIVETo explore the mechanism of anisodamine in treating infectious shock through studying effect of anisodamine on endotoxin lipopolysaccharide (LPS) induced expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in vascular endothelial cells (EC).

METHODSHuman umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. In order to evaluate a possible contribution of the nuclear factor-kappa B (NF-kappa B) pathway on the transductive effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B binding oligonucleotides.

RESULTSLPS could significantly strengthen the expression of HUVEC PAI-1 protein and TF activity and its mRNA, this effect of LPS could be markedly weakened after adding Anisodamine dose-dependently. Anisodamine could also completely block the LPS induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC.

CONCLUSIONThe possible mechanism of anisodamine in treating infectious shock may be through antagonizing LPS induced HUVEC TF and PAI-1 expression, and the antagonism might be, at least partially, transduced by path of NF-kappa B.

Cells, Cultured ; Culture Media, Conditioned ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Humans ; NF-kappa B ; metabolism ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Solanaceous Alkaloids ; pharmacology ; Thromboplastin ; biosynthesis ; genetics ; Umbilical Veins ; cytology

By study on the effect of anisodamine on lipopolysaccharide-induced expression of tissue factor (TF) in vascular endothelial cells (EC), the mechanism of anisodamine antithrombosis, as well as in the treatment of bacteraemic shock was investigated. Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method. TF activity was measured in the lysates of HUVEC by using a single step clotting assay. Specific mRNA expression was detected by Northern blotting. In order to evaluate a possible contribution of the nuclear factor (NF)-kappa B pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVECs and NF-kappa B-binding oligonucleotides. The results showed that treatment of HUVEC with LPS resulted in a significant increase in TF activity. Anisodamine dose-dependently inhibited LPS-induced upregulation of TF. These effects was also confirmed on the level of specific TF mRNA expression by Northern blotting. Furthermore, EMSA showed that anisodamine completely abolished LPS-induced NF-kappa B DNA binding activity in nuclear extracts from HUVECs treated with LPS together with anisodamine. The results suggest that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced TF expression in these cells. Its interference with the NF-kappa B pathway might--at least in part--contribute to this effect. The ability of anisodamine to counteract LPS effect on endothelial cells might be one underlying mechanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.
Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Electrophoretic Mobility Shift Assay ; Endothelium, Vascular ; metabolism ; Humans ; Lipopolysaccharides ; NF-kappa B ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Solanaceous Alkaloids ; pharmacology ; Thromboplastin ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo study the effect and mechanism of berbamine on the apoptosis of multidrug resistant leukemia K562/Adr cells and in reversing the drug resistance.

METHODSIC50 value of K562/Adr cell was determined with MTT method, cell apoptosis rate was analyzed by flow cytometry with Annexin V FITC-PI assay, with the peak and cell cycle detected by PI staining. At the same time, flow cytometry was also used in determining Caspase-3, P-GP protein expression and drug accumulating capacity in cells, and RT-PCR method was used to analyze the gene expression of mdr-1.

RESULTSBerbamine could inhibit human leukemia K562/Adr cell growth in dose-dependent manner, it could also induce cell apoptosis, increase the protein expression of Caspase-3 and the drug excretion capacity of cells, reduce the mRNA and protein expression levels of mdr-1 gene.

CONCLUSIONBerbamine could activate Caspase-3 to induce human leukemia K562/Adr cell apoptosis, and by reducing mdr-1 gene expression to reverse its multidrug resistance.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Alkaloids ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Benzylisoquinolines ; pharmacology ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Humans ; K562 Cells ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo determine effects of berbamine on the growth of leukemia cell line NB4 and explore its possible mechanisms.

METHODSThe growth of NB4 cells was examined with MTT assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in NB4 cells, and the apoptosis rate was measured by flow cytometry. The PML/RAR alpha mRNA was determined by nested-PCR, and the Survivin mRNA was tested by RT-PCR. The expression of caspase 3 protein in NB4 cells was evaluated by flow cytometry.

RESULTThe growth of NB4 cells was inhibited significantly after treated with berbamine at different concentrations for different time points, the IC(50)value was 3.860 microg/ml at 48 hours. Morphology analysis showed the characteristics of apoptosis, and the DNA agarose electrophoresis showed the typical DNA ladder. The apoptosis rate increased from 2.83% to 58.44% after treated with berbamine at 12 microg/ml for 48 hours. The expression of PML/RAR alpha mRNA presented no significant changes, however, Survivin mRNA was decreased dramatically. The protein expression of Caspase 3 increased significantly from 2.06% to 70.89% after treated with berberine at a concentration of 12 mug/ml for 48 hours.

CONCLUSIONBerbamine could inhibit the growth of leukemia cell line NB4. The induction of cell apoptosis may be one of the mechanisms for suppressing the growth of leukemia cell line NB4. Inhibition of Survivin mRNA and upregulation of Caspase 3 protein might be also involved in cell apoptosis.

Alkaloids ; pharmacology ; Apoptosis ; drug effects ; Benzylisoquinolines ; pharmacology ; Caspase 3 ; biosynthesis ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Tumor Cells, Cultured

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

Alanine Transaminase ; blood ; Alkaloids ; pharmacology ; Animals ; Aspartate Aminotransferases ; blood ; Carbon Tetrachloride ; Interleukin-10 ; analysis ; biosynthesis ; Liver Cirrhosis, Experimental ; drug therapy ; immunology ; Male ; Quinolizines ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; analysis ; biosynthesis