Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Effect of parental feeding activity on squab-induced crop sac growth in ring doves (Streptopelia risoria)

Effects of parental regurgitation feeding activity on crop sac development were studied in mate-separated male and female ring doves given 2 hr of daily exposure to food-deprived or recently fed squabs, for 4 consecutive days during the early posthatching period of the breeding cycle. In both sexes, food-deprived squabs stimulated more squab-directed activity, more parental regurgitation feeding activity, and greater crop sac development than recently fed young. Crop sac weights of males in both groups tended to be positively correlated with one or more parental activities. Correlations obtained in males exposed to food-deprived young further suggested that tactile stimuli associated with regurgitation behavior may promote crop sac development. In contrast to males, crop sac weights of females in both groups were not highly correlated with any type of contact-related parental activity or group of activities. These results, together with previous findings, suggest that nontactile stimuli from young played some role in mediating female crop sac weight differences in the two exposure conditions.     

Plasma luteinizing hormone during the breeding cycle of the female ring dove

Plasma luteinizing hormone (LH) levels in serial blood samples of female ring doves (Streptopelia risoria) were measured by radioimmunoassay method. Our findings indicate the following pattern of LH levels: LH increases during early courtship which reaches a peak (5.43 ± 0.79 ng/ml) during the nesting period. LH declines following egg-laying (3.77 ± 0.33 ng/ml) and again after hatching (2.23 ± 0.28 ng/ml). It remains high in females which laid infertile eggs initially and subsequently showed a further laying. The results are compared with published data on plasma estrogens and progesterone in the dove's breeding cycle.     

Gastrodin Alleviates Cerebral Ischaemia/Reperfusion Injury by Inhibiting Pyroptosis by Regulating the lncRNA NEAT1/miR-22-3p Axis

Cerebral ischaemia/reperfusion (I/R) injury-induced irreversible brain injury is a major cause of mortality and functional impairment in ageing people. Gastrodin (GAS), derived from the traditional Chinese herbal medicine Tianma, has been reported to inhibit the progression of stroke, but the mechanism whereby GAS modulates the progression of cerebral I/R remains unclear. The middle cerebral artery occlusion method was used as a model of I/R in vivo. Rats were pretreated with GAS by intraperitoneal injection 7 days before I/R surgery and were then treated with GAS for 7 days after I/R surgery. Additionally, an oxygen–glucose deprivation/reoxygenation model using neuronal cells was established in vitro to simulate I/R injury. 2,3,5-Triphenyltetrazolium chloride and Nissl staining were used to evaluate infarct size and neuronal damage, respectively. Lactate dehydrogenase release and cell counting kit-8 assays were used to assess neuronal cell viability. Enzyme-linked immunosorbent assay, qPCR, flow cytometry and western blotting were performed to analyse the expression levels of inflammatory factors (IL-1β, IL-18), lncRNA NEAT1, miR-22-3p, NLRP3 and cleaved caspase-1. Luciferase reporter experiments were performed to verify the association between lncRNA NEAT1 and miR-22-3p. The results indicated that GAS could significantly improve the neurological scores of rats and reduce the area of cerebral infarction. Meanwhile, GAS inhibited pyroptosis by downregulating NLRP3, inflammatory factors (IL-1β, IL-18) and cleaved caspase-1. In addition, GAS attenuated I/R-induced inflammation in neuronal cells through the modulation of the lncRNA NEAT1/miR-22-3p axis. GAS significantly attenuated cerebral I/R injury via modulation of the lncRNA NEAT1/miR-22-3p axis. Thus, GAS might serve as a new agent for the treatment of cerebral I/R injury.

     

Electrostatic control and chloride regulation of the fast gating of ClC-0 chloride channels

The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.     

Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein

Recombinant tilapia (Oreochromis mossambicus) fish metallothionein (MT) was used as a surface biosorbent for mercury removal in Escherichia coli. Fish MT conferred better resistance than did mouse or human MT. When tilapia MT (tMT) was fused with an outer-membrane protein, outer membrane protein C (OmpC), the membrane-targeted fusion protein, OmpC–tMT, gave enhanced resistance compared with cytoplasmic tMT expressed in the same host cell. The cytoplasmically expressed tMT showed high mercury adsorption (4.3 ± 0.4 mg/g cell dry weight). The cell surface that expressed E. coli showed about 25% higher adsorption ability (5.6 ± 0.4 mg/g) than the cells expressing cytoplasmic MT, attaining almost twice the level of adsorption of the control plasmid (3.0 ± 0.4 mg/g). As MTs are also known for their ability to scavenge hydroxyl-free radicals, it was also shown that tMT exhibited better radical-scavenging activities than glutathione. These results suggest that fish MT has potential for the development of a bioremediation system for mercury removal that protects the harboring E. coli host by free-radical scavenging.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

应用免疫和质谱法对亚心形扁藻氢酶蛋白进行亚细胞定位和分类

亚心形扁藻(Platymonas subcordiformis)是新发现的一株产氢海洋单细胞绿藻,经过胁迫调控可实现一定时间的持续产氢。氢酶是亚心形扁藻在胁迫条件下进行光合产氢的一个关键酶。但到目前为止,亚心形扁藻氢酶相关信息仍不清楚。利用蛋白合成抑制剂氯霉素和放线菌酮对亚心形扁藻氢酶活性进行考察,同时利用免疫印迹技术和免疫胶体金电镜对亚心形扁藻氢酶蛋白进行亚细胞定位分析。结果表明:亚心形扁藻氢酶蛋白可能由胞浆内合成,在叶绿体行使功能。采用免疫共沉淀技术富集亚心形扁藻细胞氢酶蛋白,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对免疫共沉淀复合物进行分离,从胶中切取目的蛋白条带,胶内酶解后进行基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析,得到相应的肽指纹图谱,通过搜索数据库检索初步断定亚心形扁藻氢酶蛋白为铁氢酶。     

红蚁净防治红火蚁的药效试验

以含1%氟虫腈、植物淀粉、引诱物质及粘附剂等自制的红蚁净(Pyragne)粉剂,在室内测定红火蚁Solenopsis invicta Buren各虫态的毒力。结果显示:红蚁净粉剂对红火蚁工蚁LT50=20.106h、LT90=23.78h,对有翅繁殖蚁LT50=20.951h、LT90=25.64h;工蚁能将红蚁净传递给蛹和卵引起中毒死亡。     

Apo-E genotypes and cardiovascular diseases: a sensitivity study using cross-validatory criteria

The Apolipoprotein-E (Apo-E) gene, a gene that produces proteins which help to regulate lipid levels in the bloodstream, is of interest in the study of cardiovascular diseases. An approach to making inferences about the genetic effects of the Apo-E gene has been developed by Glickman and Gagnon (2002). The framework describes the role of genetic and risk factors on the onset ages of multiple diseases, and accounts for the possibility that an individual was censored for reasons related to the diseases of interest. The framework also allows for missing genetic information, so that subjects censored prior to genetic sampling, and therefore missing such information, may still be included in the analysis. We apply an extension to this framework to the original cohort of the Framingham Heart Study for measuring the effects of different Apo-E genotypes on the onset age of various cardiovascular disease events. In particular, we compare the fit of univariate versus multivariate onset age components to the model, whether to incorporate health covariates measured at baseline or at a point later in the study, and whether to assume a heritability model for Apo-E genotype frequencies. The results of the best fitting model are presented.