The Arabidopsis anaphase-promoting complex/cyclosome subunit 1 is critical for both female gametogenesis and embryogenesis(F).

Anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ligase, plays a critical role in cell cycle control, but the functional characterization of each subunit has not yet been completed. To investigate the function of APC1 in Arabidopsis, we analyzed four mutant alleles of APC1, and found that mutation in APC1 resulted in significantly reduced plant fertility, accumulation of cyclin B, and disrupted auxin distribution in embryos. The three mutant alleles apc1-1, apc1-2 and apc1-3 shared variable defects in female gametogenesis including degradation, abnormal nuclear number, and disrupted polarity of nuclei in the embryo sac as well as in embryogenesis, in which embryos were arrested at multiple stages. All of these defects are similar to those previously identified in apc4. The mutant apc1-4, in which the T-DNA was inserted after the transmembrane domain at the C-terminus, showed much more severe phenotypes; that is, most of the ovules were arrested at the one-nucleate female gametophyte stage (stage FG1). In the apc1 apc4 double mutants, the fertility was further reduced by one-third in apc1-1/+ apc4-1/+, and in some cases no ovules even survived in siliques of apc1-4/+ apc4-1/+. Our data thus suggest that APC1, an essential component of APC/C, plays a synergistic role with APC4 both in female gametogenesis and in embryogenesis.

Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family.

Plasma membrane intrinsic proteins (PIPs) are a subfamily of aquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 (OsPIP1-1) or OsPIP2 (OsPIP2-2) in wild-type Arabidopsis, showed enhanced tolerance to salt (100 mM of NaCl) and drought (200 mM of mannitol), but not to salt treatment of higher concentration (150 mM of NaCl). Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes.

Enhanced green fluorescence protein tracks trichosanthin in human choriocarcinoma cells as a feasible and stable reporter.

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of endogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.

Trichomislin, a novel ribosome-inactivating protein, induces apoptosis that involves mitochondria and caspase-3.

Trichomislin, a novel ribosome-inactivating protein, was cloned from the genome of Trichosanthes kirilowii Maxim. The gene was recombined to prokaryotic expression vector and the protein was purified by cation-exchange chromatography. The secondary structure of trichomislin was measured by circular-dichroism analysis and the ratios of alpha-helices and beta-sheets were calculated. Trichomislin could inhibit the synthesis of protein in rabbit reticulocyte lysate systems and its reaction mechanism was to inactivate ribosome as an rRNA N-glycosidase. Antitumor analyses indicated trichomislin induced the apoptosis and inhibited the growth of choriocarcinoma cells. Further investigation showed that trichomislin could bind to and enter choriocarcinoma cells, and then increase the caspase-3 activity in a time-dependent manner. At the same time, the concentration of cytochrome c in cytosol increased while that in mitochondria decreased. These results suggested that trichomislin induced apoptosis by releasing cytochrome c from mitochondria which then triggered the caspase family member activation.

Safety assessment for genetically modified sweet pepper and tomato.

The coat protein (CP) gene of cucumber mosaic virus (CMV) was cloned from a Chinese CMV isolate, the CaMV promoter and NOS terminator added and the gene construct was transformed into both sweet pepper and tomato plants to confer resistance to CMV. Safety assessments of these genetically modified (GM) plants were conducted. It was found that these two GM products showed no genotoxicity either in vitro or in vivo by the micronucleus test, sperm aberration test and Ames test. Animal feeding studies showed no significant differences in growth, body weight gain, food consumption, hematology, blood biochemical indices, organ weights and histopathology between rats or mice of either sex fed with either GM sweet pepper or tomato diets compared with those with non-GM diets. These results demonstrate that the CMV-resistant sweet pepper and tomato are comparable to the non-GM counterparts in terms of food safety.

[Callus induction and regeneration from mature seeds of indica rice minghui 63 and anti-fungal assay of transgenic rice plants].

A large number of callus from mature seeds of indica rice minghui 63 were obtained through pre-induction on medium with 2 mg/L 2,4-D but without inorganic and organic components for 9 days. Trichosanthin gene was transferred into indica rice minghui 63 by using agrobacterium with the help of bombardment and the transgenic plants were obtained by inducing regeneration. Southern and Western blot analysis showed that the trichosanthin gene had been transferred into genome of minghui 63 and expressed in rice plants. The anti-fungal assay suggested that transgenic rice plants enhanced resistance to infection of Pyricularia oryzae.

Sequence-specific assignments of proton NMR resonance peaks and analysis of secondary structural elements of LC1, a novel antibacterial polypeptide.

LC1 is a type of novel antibacterial polypeptide secreted by a Bacillus subtilis strain. It consists of 47 residues. Using bioengineering, LC1 was expressed in E.coli DH5alpha by using recombinant plasmid PBVAB16. By means of two-dimensional DQF-COSY, TOCSY and NOESY spectroscopies, protons of all 47 residues are identified. The studies show that the secondary structures of LC1 are principally anti-parallel beta sheets and extended conformations. It was speculated that there may be a hydrophobic core around Trp(23) in its three-dimensional structure.

Capillary electrophoresis and circular dichroism study of trichosanthin and its mutants.

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-human-immunodeficiency-virus (anti-HIV). In this study, circular dichroism (CD) and capillary electrophoresis were used for the first time to study TCS and its two TCS mutants of Y55G TCS (tyrosine 55 converted to glycine) and FYY140-142GSA TCS (tripeptide phenylalanine-tyrosine-tyrosine 140-142 converted to glycine-serine-alanine). The results indicated that the substitution of amino acids changed the secondary structures and the hydrophobility of TCS. Moreover, both Y55G TCS and FYY140-142GSA TCS demonstrated attenuated cytotoxicity and reactive oxygen species (ROS) production in human choriocarcinoma cells (JAR cells) as compared to natural TCS and wild-type TCS. Our results demonstrated the cytotoxicity of TCS on JAR cells and TCS-induced production of ROS might be TCS-conformational related, suggesting that CD and capillary electrophoresis study might throw new insight into the anti-tumor and anti-HIV mechanism of TCS.