The Investigation on Polyion Complex Micelles Composed of Diammonium Glycyrrhizinate/Poly(Ethylene Glycol)–Glycidyltrimethylammonium Chloride-Grafted Polyasparthydrazide

To prepare stable polyion complex (PIC) micelles, polyasparthydrazide (PAHy) modified with glycidyltrimethylammonium groups and methoxy poly(ethylene glycol) (mPEG) (mPEG-g-PAHy-GTA) was synthesized. The cytotoxicity of the polymer was evaluated by the methyl tetrazolium assay. The polymer entrapped the diammonium glycyrrhizinate (DG) and formed polyion complexes. The effect of pH value, grafting degree of mPEG, copolymer and drug concentration on the micelle formation was investigated by means of measuring entrapment efficiency and micelle size. In vitro DG release from the PIC micelles was detected by dialysis in various media of different ionic strengths. To examine the pharmacokinetic behavior of micelles in vivo, the time course of the drug in plasma was evaluated. The cytotoxicity of the polymer was very low. The results showed that entrapment efficiency can reach about 93%, and the mean particle size was almost 50 nm. The drug release rate decreased with a decrease in ionic strength of the release medium or an increase in the PEG grafting degree. Compared with DG solution, the AUC of DG micelles had a twofold increase. The smaller clearance and longer mean residence time of the DG micelles group compared with DG solution group showed that the DG loaded in PIC micelles can reduce drug elimination and prolong the drug residence time in the blood circulation. The results indicated that PIC micelles composed of mPEG-g-PAHy-GTA would be prospective as a drug carrier to the drugs which can be ionized in solution.KEY WORDS: diammonium glycyrrhizinate, drug delivery systems, poly(ethylene glycol)–glycidyltrimethylammonium chloride-grafted polyasparthydrazide, polyion complex micelles     

Overexpression of Jatropha curcas Defensin (JcDef) Enhances Sheath Blight Disease Resistance in Tobacco

Plant defensins are small, basic, cysteine‐rich peptides, belonging to the antimicrobial peptide superfamily, commonly found in the plant kingdom. In this study, we cloned and characterized a plant defensin gene from Jatropha curcas (JcDef). JcDef carried conserved receptor binding sites and a cysteine motif, and it was phylogenetically grouped together with defensin Ec‐AMP‐D2‐like in Elaeis guineensis. JcDef is localized to cytoplasm and highly expressed in young tissues with fast metabolism such as cotyledons and stem apexes. Transgenic expression of JcDef in tobacco showed enhanced resistance against sheath blight disease caused by R. solani, indicating the antibacterial function.     

氯喹对戊四氮致痫大鼠皮质和海马白细胞介素1β及肿瘤坏死因子α表达的影响

目的观察氯喹对戊四氮致痫大鼠皮质和海马白细胞介素1β(IL-1β)及肿瘤坏死因子α(TNF-α)表达的影响,探讨其在癫痫发生发展过程中的作用.方法 48只健康雄性SD大鼠随机分为对照组(12只)、戊四氮(PTZ)致痫组(18只,60mg/kg,i.p.)和氯喹干预组(18只,氯喹0.61mg/kg,i.c.v.,2h后注射PTZ).每组确定6个时间点:1h、2h、4h、8h、12h和24h.观察大鼠行为表现,记录脑电改变,用免疫组化检测皮质和海马IL-1β和TNF-α表达的变化.结果对照组无痫样发作和痫样放电,戊四氮致痫组痫样发作重(Ⅲ-Ⅴ级),氯喹干预组轻(Ⅰ-Ⅲ级)(P<0.05);脑电记录显示戊四氮致痫组呈频发高幅的痫样波,氯喹干预组痫样波幅低且缓;LI-1β和TNF-α在戊四氮致痫组皮质和海马表达强,与对照组比较差异有显著性(P<0.05),氯喹干预组与对照组比较差异无显著性(P>0.05).结论氯喹可能通过对IL-1β和TNF-α表达的抑制减轻戊四氮致痫大鼠的痫样放电和痫样发作程度.这些结果提示,氯喹在防治癫痫方面可能是理想的抗痫剂.     

The high level of wide-compatibility of variety ‘Dular’ has a complex genetic basis

Wide-compatibility varieties (WCVs) are a special class of rice germplasm that is able to produce fertile hybrids when crossed to both indica and japonica rice varieties. WCVs may differ greatly in their spectrum and level of compatibility. The objective of this study was to determine the genetic basis of wide-compatibility conferred by ‘Dular’, a landrace variety from India that has demonstrated a high level of wide-compatibility in previous studies with a broad range of indica and japonica varieties. A three-way cross (‘Balilla/Dular//Nanjing 11’) was made and the resulting F₁ population evaluated in the field for spikelet fertility. A total of 235 plants from this population was assayed individually for restriction fragment length polymorphisms (RFLPs) at 159 marker loci covering the entire rice genome at regular intervals. Quantitative trait locus (QTL) analysis identified 5 loci, located on chromosomes 1, 3, 5, 6 and 8, as having significant effects on hybrid fertility, which jointly explained 55.5% of the fertility variation in this population. The QTL on chromosome 5 ( f5) showed the largest effect on hybrid fertility, followed by those on chromosomes 6 ( f6), 3 ( f3) and 1 ( f1), with the one on chromosome 8 ( f8) having the smallest effect. Genotypes each composed of an allele from ‘Dular’ and an allele from ‘Nanjing 11’ at four ( f3, f5, f6 and f8) of the five QTLs contributed to the increase of fertility in the population. In contrast, the genotype composed of alleles from ‘Balilla’ and ‘Nanjing 11’ at the fifth locus ( f1) was in the direction of increasing fertility. Analysis of variance using marker genotypes at the five QTLs as the groups detected two interactions involving four of the five loci, a 2-locus interaction between f5 and f8 and a 3-locus interaction among f3, f5 and f6. The level of hybrid fertility is the result of complex interactions among these loci. The implication of the present findings in the utilization of the wide-compatibility of ‘Dular’ in rice breeding programs is also discussed. Received: 21 October 1997 / Accepted: 30 December 1997     

Progesterone and DNA damage encourage uterine cell proliferation and decidualization through up-regulating ribonucleotide reductase 2 expression during early pregnancy in mice

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.     

CMTM6, the newly identified PD-L1 regulator,correlates with PD-L1 expression in lung cancers

Modulation of Immune check point regulators, especially the PD-1/PD-L1 axis, plays a critical role in successful management of a small proportion of lung cancer patients, but not so effective in the rest of lung cancer patients. A better understanding of immunotherapy non-responsive or resistant patients therefore warranted for future development of novel therapeutics. The newly identified regulator CMTM6 (CKLF-like MARVEL transmembrane domain containing 6) has been reported to serves as the stabilizer of PD-L1 and enhances the inhibitory effect of PD-L1 on immune system in both cell line and animal models, but its clinical relevance associated with PD-L1 is unknown and the current study is designed to address this question. The study using immunohistochemistry demonstrated that CMTM6 positivity from 15 out of 19 types of cancers with our in-house tissue microarray, and PD-L1 expression is always found only in CMTM6 positive cancers. CMTM6 and PD-L1 expression were analyzed in 81 lung cancer patient sample, and we observed that CMTM6 expression correlated with cancer histotypes and inversely correlated with cancer metastases, but not with patients’ age and gender. No PD-L1 expression was observed in negative CMTM6 samples. Higher expression PD-L1 is also associated with higher CMTM6 expression. In summary, CMTM6 expression is associated with PD-L1 expression, as well as lung cancer histotypes and metastasis. The results thus for the first time confirmed earlier reports on CMTM6/PD-L1 connection, from a clinical aspect of analysis.     

MLKL mediates apoptosis via a mutual regulation with PERK/eIF2α pathway in response to reactive oxygen species generation

The pseudokinase mixed lineage kinase domain-like protein (MLKL) is a core effector of necroptosis, and its function in necroptosis is widely studied. However, the function of MLKL in apoptosis remains unclear. In the present study, the role of MLKL in chelerythrine (CHE)-promoted apoptosis was studied. A special band of MLKL (i.e., *MLKL) was observed after treatment with CHE. MLKL and *MLKL were accumulated in the nucleus upon treatment with CHE and MLKL silencing reversed the CHE-induced apoptosis. Blockade of CHE-triggered reactive oxygen species (ROS) generation or inhibition of CHE-activated protein kinase-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2 α subunit (eIF2α) pathway reversed the apoptosis. A decreased ROS level inhibited CHE-mediated nuclear translocation of MLKL and *MLKL and the activation of eIF2α, whereas MLKL or eIF2α silencing did not affect the CHE-triggered ROS generation. Furthermore, MLKL silencing prevented the CHE-activated eIF2α signal, and eIF2α silencing blocked the CHE-induced nuclear translocation of MLKL and *MLKL. Our studies suggested that CHE possibly induces apoptosis through the nuclear translocation of MLKL and *MLKL, which is promoted by a mutual regulation between MLKL and PERK–eIF2α pathway in response to ROS formation. The present study clarified the new function of MLKL in apoptosis.     

Vascular basement membrane-derived multifunctional peptide, a novel inhibitor of angiogenesis and tumor growth

Vascular basement membrane-derived multifunctional peptide(VBMDMP)gene(fusion geneof the human immunoglobulin G3 upper hinge region and two tumstatin-derived fragments)obtained bychemical synthesis was cloned into vector pUC19,and introduced into the expression vector pGEX-4T-1 toconstruct a prokaryotic expression vector pGEX-4T-1-VBMDMP.Recombinant VBMDMP produced inEscherichia coli has been shown to have significant activity of antitumor growth and antimetastasis inLewis lung carcinoma transplanted into mouse C57B1/6.In the present study,we have studied the ability ofrVBMDMP to inhibit endothelial cell tube formation and proliferation,to induce apoptosis in vitro,and tosuppress tumor growth in vivo.The experimental results showed that rVBMDMP potently inhibited prolif-eration of human endothelial(HUVEC-12)cells and human colon cancer(SW480)cells in vitro,with noinhibition of proliferation in Chinese hamster ovary(CHO-K1)cells.rVBMDMP also significantly inhibitedhuman endothelial cell tube formation and suppressed tumor growth of SW480 cells in a mouse xenograftmodel.These results suggest that rVBMDMP is a powerful therapeutic agent for suppressing angiogenesisand tumor growth.