Proposal for the classification of sinomenine alkaloids.

The chemical structure of sinoacutine is formed by a phenanthrene nucleus and an ethylamine bridge. Because it has a similar parent structure to morphine, it is subdivided into morphinane. At present, all reports have pointed out that the basic skeleton of morphine alkaloids is salutaridine (the isomer of sinoacutine), which is generated by the phenol coupling reaction of (R)-reticuline. This study shows that the biosynthetic precursors of sinoacutine and salutaridine are different. In this paper, the sinoacutine synthetase (SinSyn) gene was cloned from Sinomenium acutum and expressed SinSyn protein. Sinoacutine was produced by SinSyn catalyzed (S)-reticuline, according to the results of enzyme-catalyzed experiments. The optical activity, nuclear magnetic resonance, and mass spectrum of sinoacutine and salutaridine were analyzed. The classification and pharmacological action of isoquinoline alkaloids were discussed. It was suggested that sinoacutine should be separated from morphinane and classified as sinomenine alkaloids.

[Identification of differentially expressed genes of acute hypoxia-treated HepG2 cells and hypoxia-acclimatized HepG2 cells].

To provide necessary information for further understanding of molecular mechanism of hypoxia acclimatization, the differentially expressed genes of HepG2 cells exposed to normoxia, acute hypoxia-treated cells which were exposed to 1% oxygen for 48 h, and hypoxia-acclimatized HepG2 cells which were cultured for 6 circles of alternate low oxygen (1% oxygen for 24 h) and normal oxygen (21% oxygen for 24 h), were identified respectively by combining the suppression subtractive hybridization (SSH) and cDNA microarray. Thirty-seven genes were expressed differentially in cells exposed to 1% oxygen for 48 h compared with those in cells exposed to normoxia. The expression of all these 37 genes was down-regulated, including the genes participating in cell cycle, cell response to stimulus, and cell signal transduction, and cell cytoskeleton formation, the genes associated with transcription and cell metabolism, 4 expressed sequence tags (ESTs), and 12 genes of which the functions are not known. There is a novel gene sequence, which has not been found in existing databases. There were only 6 genes differentially expressed in the hypoxia-acclimatized cells compared with cells exposed to normoxia, including two mitochondrion genes, metalloprotease-1 gene, ferritin gene, thymosin beta-4 and TPT1 genes. The expressions of mitochondrion ND4, ferritin, thymosin beta-4 and TPT1 were up-regulated, while the expressions of mitochondrion ND1 gene and metalloproease-1 gene were down-regulated. Cell tolerance to hypoxia increased after the cells were hypoxia-acclimatized. The different gene expression patterns of the acute hypoxia-treated cells and the hypoxia-acclimatized cells may be related to the increased tolerance of the cells to hypoxia.

[Effects of acute hypoxia and intermittent hypoxic acclimatization on erythropoietin and hypoxia-inducible factor-1alpha gene expression in rat hepatic and renal tissues].

OBJECTIVE: To explore the effects of acute hypoxia and intermittent hypoxic acclimatization on gene expression of erythropoietin (EPO) and hypoxia-inducible factor-1alpha (HIF-1alpha) in rat hepatic and renal tissues. METHODS: Twenty-four rats (weight was 180 to 220 g) were divided into three groups: normal control group (NC), intermittent hypoxic acclimatization (IH) and acute hypoxia groups (AH). Gene expression of EPO and HIF-1alpha were examined with Northern dot blot method. RESULTS: As compared with NC group, the levels of EPO gene expression in rat hepatic and renal tissues in AH group were both significantly elevated (P<0.05 or P<0.01). In IH group, the contents of EPO mRNA in hepatic and renal tissues were apparently decreased versus AH group (P<0.01), whilst were not differently elevated in comparison with group NC (P>0.05). In hepatic tissue from AH group, the content of HIF-1alpha mRNA were more than those in NC group and IH group, while the levels of gene expression of HIF-1alpha were no substantial change in renal tissues from different groups (all P>0.05). CONCLUSION: Hypoxic acclimatization can inhibit the increment of EPO gene expression induced by acute hypoxia in rat hepatic and renal tissues, in which HIF-1alpha may play important roles.

Structural requirement of spirostanol glycosides for rat uterine contractility and mode of their synergism.

OBJECTIVES: Total steroidal saponins extracted from the rhizome of Paris polyphylla (TSSP) have been used in China for the treatment of abnormal uterine bleeding. The aim of this study was to analyse the structure-activity relationship of steroidal saponins purified from P. polyphylla Sm. var. yunnanensis on rat myometrial contractions, and investigate the synergism among themselves as well as with known inherent agonists, such as Prostaglandin F(2alpha) (PGF-2alpha). METHODS: In this study, 22 steroidal saponins purified from TSSP were screened for their contractile activity in isolated uterine strips from estrogen-primed rats. KEY FINDINGS: It was shown that spirostanol glycosides exhibited inducible or inhibitory activity in rat uterine contraction based on the difference of their structures, which was not only attributed in part to the number, the length and the position of sugar side chains attached by a glycoside, but also related to the structure of the aglycone. Furthermore, synergistic actions were observed among pennogenin or diosgenin glycosides as well as with the known inherent agonist PGF-2alpha, indicating they may share, at least in part, similar pathways with PGF-2alpha in stimulating myometrial contractions. Finally, the contractile response of rat myometrium to spirostanol glycosides was significantly enhanced with advancing pregnancy. CONCLUSIONS: Together, these data support the possibility that some spirostanol glycosides may represent a new type of contractile agonist for the uterus and their synergism may be responsible for the therapeutic effect of TSSP on abnormal uterine bleeding.

Melissoidesin G, a diterpenoid purified from Isodon melissoides, induces leukemic-cell apoptosis through induction of redox imbalance and exhibits synergy with other anticancer agents.

Melissoidesin G (MOG) is a new diterpenoid purified from Isodon melissoides, a plant used in Chinese traditional medicine as antitumor and anti-inflammatory agents. In our study, MOG was shown to specifically inhibit the growth of human leukemia cell lines and primary acute myeloid leukemia (AML) blasts via induction of apoptosis, with the evidence of mitochondrial DeltaPsim loss, reactive oxygen species production, caspases activation and nuclear fragmentation. Furthermore, it was shown that thiol-containing antioxidants completely blocked MOG-induced mitochondrial DeltaPsim loss and subsequent cell apoptosis, while the inhibition of apoptosis by benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone only partially attenuated mitochondrial DeltaPsim loss, indicating that MOG-induced redox imbalance is an early event upstream to mitochondrial DeltaPsim loss and caspase-3 activation. Consistently, it was found that MOG rapidly decreased the intracellular glutathione (GSH) content in a dose-dependent manner and the significance of GSH depletion in MOG-induced apoptosis was further supported by the protective effects of tert-butylhydroquinone (tBHQ) and the facilitative effects of DL-buthionine (S,R)-sulfoximine (BSO). Furthermore, it was showed that GSH depletion induced by MOG rendered some leukemia cell lines more sensitive to arsenic trioxide (As2O3), doxorubicin or cisplatin. Additionally, the synergistic apoptotic effects of MOG with As2O3 were detected in HL-60 and primary AML cells, but not in normal cells, suggesting the selective toxicity of their combination to the malignant cells. Together, we proposed that MOG alone or administered with other anticancer agents may provide a novel therapeutic strategy for leukemia.

[Effects of acute hypoxia and intermittent hypoxic acclimatization on vascular endothelial growth factor gene expression in HepG2 cells].

AIM: To explore the influence of acute hypoxia and intermittent hypoxic acclimatization on vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha (HIF-1alpha) gene expression in HepG2 cells underlying their possible biological significance. METHODS: HepG2 was cultured in 1% O2 for 24 hours, then in 21% O2 for another 24 hours, which composed a hypoxic exposure cycle. After 6 cycles, HepG2 cells reached the status of hypoxic acclimatization. Gene transcription and translation of VEGF and HIF-1alpha were detected with Northern blot and Western blot methods. RESULTS: Acute hypoxia could induce gene transcription and translation of VEGF and HIF-1alpha. After intermittent hypoxia acclimatization, the contents of VEGF and HIF-1alpha mRNA were 108.6% +/- 17.7% and 116.7% +/- 19.8% of those in normoxic control cells, while the protein contents were significantly increased to 1.4 and 2.7 times of those in control cells, respectively (P < 0.05). The protein expression levels of VEGF and HIF-1alpha were decreased in cells subjected to hypoxia acclimatization compared to cells treated with acute hypoxia. CONCLUSION: When HepG2 cells reached the status of hypoxic acclimatization, the acute hypoxia-induced increment of VEGF gene transcription and translation in cells were inhibited, in which HIF-1alpha might play an important role.

Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida.

The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eisenia foetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The cDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.