Taxa-4(5),11(12)-diene is the precursor for paclitaxel biosynthesis. The diterpenoid paclitaxel (marketed as Taxol), a plant secondary metabolite isolated from yew, is an effective drug widely used in the treatment of numerous cancers. However, further application of taxol has been restricted due to its low yield in plants and the difficulties in extraction. To increase the intact isoprene flux, we constructed the fusion gene plasmid pBgGGTS and individual cassette plasmid pBgGGgTS to enhance the expression levels of geranylgeranyl diphosphate synthase gene (ggpps) and a taxadiene synthase gene (ts) in Coprinopsis cinerea. These two plasmids were separately transformed into C. cinerea LT2 strain, resulting in several putative transformants. Putative transformants were determined by PCR technique, indicating that 5 out of 13 putative transformants transformed by pBgGGTS and 6 out of 13 putative transformants transformed by pBgGGgTS, respectively. Additionally, the Southern blotting analysis of these 10 transformants confirmed that both ggpps and ts gene were stably integrated into the genome of C. cinerea. Crude extracts from each of the transformants were analyzed. There is no difference in the mycelium extracts among the wild-type LT2 and two types of transformants. However, analysis of culture filtrates indicated that an additional GC peak was found at the retention time of 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. According to peak area, the amounts of taxa-4(5),11(12)-diene in each fermented broth were 44 ng/L (transformed with pBgGGgTS) and 30 ng/L (transformed with pBgGGTS), respectively. In conclusion, co-expression of the ggpps and ts gene could increase the taxadiene production in C. cinerea.
Agaricales ; metabolism ; Alkenes ; metabolism ; Diterpenes ; metabolism ; Farnesyltranstransferase ; genetics ; metabolism ; Genetic Engineering ; Isomerases ; genetics ; metabolism ; Paclitaxel ; Plasmids

BACKGROUND: Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The extracellular-signal-regulated kinase (ERK) signaling pathway plays a crucial role in almost all cell functions, including proliferation, differentiation, survival, and death. However, the effect of ERK inhibition on oxysterol-induced apoptosis remains uncertain. METHODS: This study assessed the effect of ERK inhibition on the apoptotic effect of 7-ketocholesterol. RESULTS: Treatment with 7-ketocholesterol increased phosphorylated-ERK1/2 levels in differentiated PC12 cells, while the total amount of ERK was not altered. 7-Ketocholesterol decreased Bid and Bcl-2 levels, increased Bax and p53 levels, and promoted cytochrome c release, which elicits the activation of caspases (-8, -9, and -3), nuclear damage, and cell death. ERK and farnesyltransferase inhibitors inhibited the 7-ketocholesterol-induced phosphorylation of ERK1/2, activation of apoptosis-related proteins, and cell death in PC12 cells. CONCLUSIONS: The ERK and farnesyltransferase inhibitors, which did not exhibit toxicity, may inhibit the 7-ketocholesterol toxicity on differentiated PC12 cells by suppressing the activation of the caspase-8-dependent pathway as well as activation of the mitochondria-mediated cell-death pathway, leading to the activation of caspases. The inhibition of ERK may confer a beneficial protective effect against the neuronal cell injury induced by cholesterol oxidation products.
Animals ; Apoptosis ; Caspases ; Cell Death ; Cholesterol ; Cytochromes c ; Farnesyltranstransferase ; Ketocholesterols ; Neurons ; PC12 Cells ; Phosphorylation ; Phosphotransferases ; Proteins

β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production.
Basidiomycota ; enzymology ; Farnesyltranstransferase ; genetics ; metabolism ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; Metabolic Engineering ; Polyisoprenyl Phosphates ; Saccharomyces cerevisiae ; metabolism ; beta Carotene ; biosynthesis

A full-length cDNA of GGPPS gene from Tripterygium wilfordii suspension cells was obtained by use of RACE strategy (GeneBank: KM978333), and then analyzed by bioinformatics approaches. TwGGPPS cDNA has 1857 nucleotides and an open reading frame (ORF) encoding a protein of 514 amino acid residues. The deduced protein has isoelectric point (pI) of 7.85, a calculated molecular weight about 57.13 kD, 5 conserved domains and 2 functional domains. PSORT Prediction showed it was located at plasma membrane. Phylogenetic analysis demonstrated that TwGGPPS1 was similar to GGPPS from other species of plants. For the first time the cloning of geranylgeranyl diphosphate synthase gene from T. wilfordii was reported, it lays the foundation for further research of diterpenoids biosynthetic pathway.
Amino Acid Sequence ; Cloning, Molecular ; Farnesyltranstransferase ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Tripterygium ; chemistry ; enzymology ; genetics

AIMDesign, synthesis and evaluation of a series of 7-imidazolylalkanamido-1-carboxylalkylbenzodiazepine farnesyltransferase (FTase) inhibitors.

METHODS AND RESULTSCoupling of imidazolylalkylcarboxylic acids and 1-substituted 7-aminobenzodiazepines (5a-5c) yielded 10 new compounds (6-12, 16-18) which were biologically tested against FTase using scintillation proximity assay method.

CONCLUSIONFive target compounds were found to be potential farnesyltransferase inhibitors.

Alkyl and Aryl Transferases ; antagonists & inhibitors ; drug effects ; Benzodiazepines ; chemical synthesis ; chemistry ; pharmacology ; Farnesyltranstransferase ; Imidazoles ; chemical synthesis ; chemistry ; pharmacology ; Inhibitory Concentration 50 ; Molecular Conformation ; Molecular Structure ; Structure-Activity Relationship

Recent years, the incidence and mortality of prostate cancer have increased dramatically in China. At earlier stages, most diagnosed prostate cancers are responsive to androgen depletion treatment, yet, nearly all patients will eventually progress to metastatic androgen-independent prostate cancer (AIPC), which still has no effective therapeutic method or drug to deal with. 11'-Deoxyverticillin A (C42) belongs to the family of epipolythiodioxopiperazines (ETPs), an interesting class of fungal toxins that inhibit farnesyl transferase. Compounds holding such a property have been explored as putative anticancer agents. In this study, using PC3M cells, an AIPC cell line, we investigated the effect of the compound on apoptosis and explored the underlying mechanism. It revealed that C42 markedly enhanced the activity of caspase-3/7 and increased the accumulation of the cleaved PARP, all of which are the markers of apoptosis. It also revealed that C42 either decreased cell viability or inhibited the growth of PC3M cells. Moreover, we observed that the loss of cell viability and cell growth inhibition induced by C42 were both time- and dosage dependent. Taken together, we indicated that C42 can induce caspase-dependent apoptosis in AIPC cells, and the results presented here will broaden our knowledge about the molecular mechanisms by which C42 exerts its anticancer activity, and future work in this direction may provide valuable information in the development of these compounds into effective cancer therapeutic strategies against androgen-independent prostate cancer.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 7 ; metabolism ; Cell Line, Tumor ; Disulfides ; pharmacology ; Farnesyltranstransferase ; antagonists & inhibitors ; Humans ; Male ; Mycotoxins ; pharmacology ; Piperazines ; pharmacology ; Prostatic Neoplasms ; pathology

BACKGROUNDPrimary liver cancer (PLC) is a common malignant tumor. Over the past decade, although farnesyltransferase (FTase) has emerged as a significant target for anticancer therapies and has become a hotspot of cancer research, its exact mechanism of action remains unknown. The aim of this study was to investigate the expression of FTase in PLC and its role in the development of PLC.

METHODSExpression of FTase was detected by real-time fluorescent quantitative-polymerase chain reaction (FQ-PCR) in cancer and surrounding normal tissues from 32 patients with PLC.

RESULTSExpression of FTase mRNA in PLC was significantly higher than that in normal hepatic tissues (P < 0.001). Overexpression of FTase was as high as 87.5%. The positive rate for FTase mRNA in the high tendency to metastatic recurrence group was obviously higher than that in the low tendency to metastatic recurrence group (P = 0.02). The positive rate for FTase mRNA in patients with metastatic recurrence during postoperative follow-up was also significantly higher than that in those without metastatic recurrence (P = 0.01).

CONCLUSIONSThe level of FTase mRNA expression in cancer tissues is much higher than in normal tissues. FTase may play an important role in the genesis and development of PLC and may be one of the reliable markers for the metastatic activity gained by liver tumor cells. FTase could be used clinically in predicting metastatic recurrence of PLC.

Adult ; Aged ; Farnesyltranstransferase ; genetics ; Female ; Humans ; In Vitro Techniques ; Liver Neoplasms ; enzymology ; pathology ; Male ; Middle Aged ; Polymerase Chain Reaction ; RNA, Messenger ; Young Adult

PURPOSE: Genetic factor is an important predisposing element influencing the susceptibility to osteoporosis and related complications. The purpose of the present study is to investigate whether genetic polymorphisms of farnesyl diphosphate synthase (FDPS) or geranylgeranyl diphosphate synthase (GGPS) genes were associated with the response to bisphosphonate therapy. MATERIALS AND METHODS: In the present study, 144 Korean women with osteoporosis were included. Among 13 genetic polymorphisms found within the FDPS and GGPS1 gene, 4 genetic polymorphisms with frequencies > 5% were selected for further study. Bone mineral density (BMD) response after 1 year treatment of bisphosphonate therapy was analyzed according to the genotypes. RESULTS: Women with 2 deletion allele of GGPS1 -8188A ins/del (rs3840452) had significantly higher femoral neck BMD at baseline compared with those with one or no deletion allele (0.768 +/- 0.127 vs. 0.695 +/- 0.090 respectively; p = 0.041). The response rate of women with 2 deletion allele of GGPS1 -8188A ins/del (28.6%) was significantly lower than the rate of women with one (81.4%) or no deletion allele (75.0%) (p = 0.011). Women with 2 deletion allele of GGPS1 -8188A ins/del had 7-fold higher risk of non-response to bisphosphonate therapy compared with women with other genotypes in GGPS1 -8188 after adjusting for baseline BMD (OR = 7.48; 95% CI = 1.32-42.30; p = 0.023). Other polymorphisms in FDPS or GGPS1 were not associated with lumbar spine BMD or femoral neck BMD. CONCLUSION: Our study suggested that GGPS1 - 8188A ins/del polymorphism may confer susceptibility to femoral neck BMD response to bisphosphonate therapy in Korean women. However, further study should be done to confirm the results in a larger population.
Aged ; Asian Continental Ancestry Group ; Bone Density/*drug effects/*genetics ; Bone Density Conservation Agents/*pharmacology ; Dimethylallyltranstransferase/*genetics ; Diphosphonates/*pharmacology ; Farnesyltranstransferase/*genetics ; Female ; Geranyltranstransferase/*genetics ; Humans ; Middle Aged ; Polymorphism, Genetic/*genetics

The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (DeltaPsim) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.
Antimalarials/*pharmacology ; Enzyme Inhibitors/*pharmacology ; Farnesyltranstransferase/*antagonists & inhibitors/genetics/*metabolism ; Humans ; Malaria, Falciparum/drug therapy/*parasitology ; Mitochondria/*drug effects/metabolism ; Plasmodium falciparum/drug effects/*enzymology/genetics ; Protozoan Proteins/*antagonists & inhibitors/genetics/metabolism ; Quinolones/*pharmacology

Ras/Raf/MEK/ERK singal transduction plays an important role in cell proliferation, differentiation, apoptosis, metastasis and metabolism. This investigation focused on this signal pathway and chose farnesyl transferase (FTase) as the main target and Raf-1 kinase as the second target. A lot of compounds were selected to construct the pharmacophore models of farnesyl transferase inhibitors (FTIs) and Raf-1 kinase inhibitors by using computer-aided drug design (CADD). The pharmacophore of FTIs is constituted by a hydrogen bonding acceptor, an aromatic ring, a positive ionizable and two hydrophobic regions; the pharmacophore of Raf-1 kinase is constituted by a hydrogen donor, a hydrogen acceptor, a hydrophobic regions and an aromatic ring. There are some similarities between the two pharmacophores. After analysis of the constructions of these two pharmacophores, some new aminomethylbenzoic acid derivatives with good forecasting activity against both of FTase and Raf-1 kinase were designed with these new pharmacophore models.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Computer-Aided Design ; Drug Delivery Systems ; Drug Design ; Enzyme Inhibitors ; chemical synthesis ; chemistry ; Farnesyltranstransferase ; antagonists & inhibitors ; Molecular Structure ; Proto-Oncogene Proteins c-raf ; antagonists & inhibitors ; Signal Transduction