化学调控剂对水稻不育系育性恢复效应的研究

１９９２年３月至１１月,我们在海南和长沙两地,先后使用３８种化学调控剂,对水稻两用不育系,三系不育系及普通核不育系进行了喷施和注射试验,筛选出７种较为有效的调控剂,代号为ＣＲ１—ＣＲ７,其中ＣＲ５和ＣＲ７表现较为突出。这些调控剂对两用不育系和三系不育系均表现出一定程度的恢复效应,且ＣＲ１和ＣＲ２还能在可育条件下提高培矮６４Ｓ的结实率,但所有调控剂对普通核不育系均无恢复作用。     

Targeted Exosomes for Drug Delivery: Biomanufacturing,Surface Tagging,and Validation

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer‐targeted exosomes for drug delivery. Specifically, a scalable, robust, high‐yield, cell line based exosome production process is created in a stirred‐tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)‐exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb‐exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb‐exosomes. The in vitro anti‐cancer toxicity study shows high cytotoxicity of mAb‐exosome‐romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti‐cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large‐scale animal studies and to facilitate the translation from research to clinics.     

Molecular characterization of avian-like H1N1 swine influenza a viruses isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-like H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3′ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.     

RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS

RILP (Rab7-interacting lysosomal protein) is a key regulator for late endosomal/lysosomal trafficking, and probably a tumor suppressor in prostate cancer. However, the role of RILP in other cancers and the underlying mechanism for RILP in regulating the invasion of cancer cells remain to be investigated. In this study, we showed that overexpression of RILP in breast cancer cells inhibits the migration and invasion, whereas the depletion of RILP by RNAi-mediated knockdown promotes the migration and invasion. We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS. Immunofluorescence microscopy revealed that RalGDS can be recruited to the late endosomal compartments by RILP. Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS. Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.Diverse alternations of oncogenic factors can either activate or inactivate signaling pathways involved in cell proliferation, migration and apoptosis that are intimately associated with cancer development.^{1, 2, 3} Recent studies suggest that the derailed membrane trafficking is also closely related to cancer development. Activation or attenuation of signal transduction is usually linked to membrane trafficking. The recycling and degradation of surface receptors, such as EGFR, will influence downstream signaling pathways.^{4, 5} Therefore, the cross-talk between membrane trafficking and signaling pathway could be the novel mechanism associated with cancer development.Alternations of the membrane trafficking machineries are established as the causes for some cancers. For examples, Rab25 is overexpressed in breast and ovary caners,⁶ and recent investigations suggest that Rab25 is also related to other cancers.^{7, 8, 9} Arf6 is a vital regulator for the invasive activity of breast cancer cells.¹⁰ Disordered membrane trafficking is emerging as an important property during tumorigenesis, thus the membrane trafficking machineries are potential therapeutic targets for cancer treatment.Rab small GTPases are considered as the master regulators for membrane trafficking.¹¹ The interactions between Rab proteins and their downstream effectors are involved in various steps of vesicle trafficking such as tethering and fusion. Aberrant activities of Rab proteins are closely related to some cancers.^{12, 13, 14, 15} Some Rab proteins mediate the trafficking of cargos, especially membrane proteins on the plasma membrane, such as integrin and E-cadherin. Their aberrant trafficking is proposed to be the underlying mechanism for the functional regulation of Rab protein in cancer cells.^{16, 17}Rab7, together with its downstream effector RILP (Rab7-interacting lysosomal protein), are the key regulators for late endosomal/lysosomal trafficking. RILP interacts with activated GTP-bound Rab7 through its carboxylic terminal region, whereas interacting with dynein/dynactin complex is mediated through its amino region, driving late endosomal/lysosomal trafficking, especially lysosomal positioning.^{18, 19} Rab7 has been demonstrated to be an important factor for cell growth and survival.^{20, 21} Recently, Steffan et al.²² found that RILP suppresses the invasion of prostate cancer cells through inhibiting the anterograde trafficking of lysosomes.²³ Whether the potential role of Rab7-RILP in cell migration/invasion is also implicated in other cancers is of interest to investigate and the underlying molecular mechanism is yet to be defined.In this study, we found that RILP suppresses the proliferation, migration and invasion of breast cancer cells. We also identified (Ral guanine nucleotide dissociation stimulator (RalGDS) as a novel interacting partner for RILP. The interaction of RILP with RalGDS modulates the activity of RalA. Our results suggest that RILP suppresses the invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS.     

Selenite and ebselen supplementation attenuates <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in d-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), β-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that d-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and β-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.     

EGF receptor clustering is induced by a 0.4 mT power frequency magnetic field and blocked by the EGF receptor tyrosine kinase inhibitor PD153035

Atomic force microscopy (AFM), transmission electron microscopy (TEM), and confocal laser scanning microscopy were used to investigate the effects of a 50 Hz 0.4 mT magnetic field (MF) on the clustering of purified epidermal growth factor receptors (EGFRs) and EGFRs in Chinese hamster lung (CHL) cell membrane. The results demonstrate that exposing purified EGFRs to the MF for 30 min induces receptor clustering. The peak height of apparent clusters increased from 1.42 +/- 0.18 (sham-exposed) to 3.08 +/- 0.38 nm (exposed) while the mean half-width increased from 21.7 +/- 2.2 to 33.0 +/- 4.0 nm. A similar effect was also observed by TEM. Treatment of purified EGFR with PD153035 (PD), an EGFR-specific tyrosine kinase (TK) inhibitor, inhibited the MF-induced EGFR clustering of the purified proteins, an effect also observed for the receptors in cell membrane in the absence of EGF. These results strongly suggest that the 50 Hz 0.4 mT MF interferes with the EGFR signaling pathway, most likely by interacting with the cytoplasmic TK domain.     

LC-MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies

Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC-MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor→product ion transitions were monitored at m/z 282.8→120.9 for helicid and m/z 326.9→192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2-20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24h, 4°C for at least 24h, -20°C for at least 1 month, and for routine three freeze-thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.