DAB致大鼠肝癌过程中免疫组化及免疫电镜研究

本文采用二甲氨基偶氮苯(DAB)诱发的大鼠肝癌模型,运用组织病理学、血清学、免疫组化及免疫电镜技术相结合的方法,对诱癌过程中各个不同时期肝脏的组织病理学变化及肝癌阳性标志物AFP的表达进行了动态观察。结果显示:早在血清AFP浓度上升和由卵圆细胞转变而来的小肝细胞表达AFP之前,肝小叶中就出现了散在的AFP阳性肝细胞,我们认为这种AFP阳性肝细胞可作为肝细胞癌前病变的早期征象之一。在AFP阳性的肝细胞内,AFP主要定位于核周间隙、粗面内质网和高尔基复合体。     

Localization of P2X and P2Y receptors in dorsal root ganglia of the cat.

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X(5) receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y(2) = P2Y(4)>P2X(3)>P2X(2) = P2X(7)>P2X(6)>P2X(1) = P2X(4)>P2Y(1). P2X(3), P2Y(2), and P2Y(4) receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y(2), P2X(1), P2X(3), P2X(4), and P2X(6) receptor staining was detected in small- and medium-diameter neurons. However, P2Y(4), P2X(2), and P2X(7) staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X(3) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y(4) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y(2) receptor-positive neurons also stained for IB(4), CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X(3)-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y(1) receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.     

艰难梭菌荚膜多糖单糖组成的气相色谱分析

本文对两株艰难梭菌（ＣｄＡ３１８和ＣｄＣＤＣ２７９）荚膜多糖进一步分析,结果证实纯化的艰难梭菌荚膜多糖为单一均匀的物质;经气相色谱分析表明,其单糖组分主要由Ｄ－葡萄糖、Ｄ－甘露糖和Ｄ－半乳糖组成,不同菌株的荚膜多糖可能有微小差异。     

Structural analysis and antitussive evaluation of five novel esters of verticinone and bile acids

Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation, which consists of Snake Bile (Chinese name “Shedan”) and Fritillariae Cirrhosae (Chinese name “Chuanbei”), is the most popular antitussive and expectorant formulation in Chinese communities. However, the clinical application of Shedan-Chuanbei powder is now stringently limited because of the shortage of the two crude medicinal materials, especially for the sake of animal protection. In addition, the inherent defects of the most of the complex of traditional Chinese medicine such as the indistinct basal pharmacodynamic materials and the difficulties in quality control had blocked them heading into the international medicinal market. So we attempted to seek new substitute for Shedan-Chuanbei powder for antitussive drugs. In order to gain some new compounds with better bioactivity and attenuated toxicity, we tried to combine two kinds of drugs through ester bond. Enlightened with “combination principle” in drug discovery, we synthesized five novel esters of verticinone and bile acids, both of which are the major bioactive components in Shedan-Chuanbei powder. We then evaluated the antitussive activity and the acute toxicity of the five ester-linked compounds. The five ester-linked compounds had much more potent antitussive activity and expectorant activity than single bile acids at the same doses, and had equivalent antitussive activity and expectorant activity in comparison with about double moles dose of the monomer verticinone. Especially, cholic acid–verticinone ester had much more potent antitussive effects than the monomer verticinone or cholic acid at the same dose. A further acute toxicity study showed that the LD₅₀ values of the five ester-linked compounds exceeded 3.5 g/kg by intraperitoneal injection in mice. Based on the studies of pharmacology and acute toxicity, the five ester-linked compounds have synergic pharmacodynamic action and attenuated toxicity compared with single verticinone and single bile acids.     

A Self-microemulsifying Drug Delivery System (SMEDDS) for a Novel Medicative Compound Against Depression: a Preparation and Bioavailability Study in Rats

AJS is the code name of an untitled novel medicative compound synthesized by the Tasly Holding Group Company (Tianjin, China) based on the structure of cinnamamide, which is one of the Biopharmaceutics Classification System (BCS) class II drugs. The drug has better antidepressant effect, achieved by acting on the 5-hydroxytryptamine receptor. However, the therapeutic effects of the drug are compromised due to its poor water solubility and lower bioavailability. Herein, a self-microemulsifying drug delivery system (SMEDDS) was developed to improve its solubility and oral bioavailability. AJS-SMEDDS formulation was optimized in terms of drug solubility in the excipients, droplet size, stability, and drug precipitation using a pseudo-ternary diagram. The pharmacokinetic study was performed in rats, and the drug concentration in plasma samples was assayed using the high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS) method. The optimized formulation for SMEDDS has a composition of castor oil 24.5%, Labrasol 28.6%, Cremphor EL 40.8%, and Transcutol HP 2.7% (co-surfactant). No drug precipitation or phase separation was observed from the optimized formulation after 3 months of storing at 25°C. The droplet size of microemulsion formed by the optimized formulation was 26.08 ± 1.68 nm, and the zeta potential was −2.76 mV. The oral bioavailability of AJS-SMEDDS was increased by 3.4- and 35.9-fold, respectively, compared with the solid dispersion and cyclodextrin inclusion; meanwhile, the C_max of AJS-SMEDDS was about 2- and 40-fold as great as the two controls, respectively. In summary, the present SMEDDS enhanced oral bioavailability of AJS and was a promising strategy to orally deliver the drug.KEY WORDS: bioavailability, HPLC-MS/MS, self-microemulsifying drug delivery system, solubilization, stability     

Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study

Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM₁₀ and personal PM_2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM₁₀ was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM₁₀ effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM₁₀ effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM_2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM₁₀, PM_2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM₁₀ affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.     

Increased Risk of Cutaneous Melanoma Associated with p53 Arg72Pro Polymorphism

Objective

The objective of this study was to test the hypothesis that p53 Arg72Pro polymorphism may contribute to an increased risk of cutaneous melanoma (CM).

Methods

By searching the databases of PubMed, EMBASE, and Web of Science, a total of 8 eligible case-control studies with 1,957 CM cases and 2,887 controls were included in this meta-analysis. Stata software was used to analyze all the statistical data.

Results

The pooled data by a fixed-effects model suggested an increased risk of CM associated with p53 Arg72Pro polymorphism under the genetic model of Arg/Pro vs. Pro/Pro without heterogeneity (OR_{Arg/Pro vs. Pro/Pro} = 1.76, 95% CI = 1.55-1.99, P _{heterogeneity} = 0.075). A similar trend was seen in subgroups of hospital-based studies and population-based studies.

Conclusion

Our meta-analysis based on all studies shows that the p53 Arg72Pro polymorphism may increase individual susceptibility to CM, particularly in Caucasians and could serve as a biomarker to predict the population at high risk of CM.     

A Pathway-Centric Survey of Somatic Mutations in Chinese Patients with Colorectal Carcinomas

Previous genetic studies on colorectal carcinomas (CRC) have identified multiple somatic mutations in four candidate pathways (TGF-β, Wnt, P53 and RTK-RAS pathways) on populations of European ancestry. However, it is under-studied whether other populations harbor different sets of hot-spot somatic mutations in these pathways and other oncogenes. In this study, to evaluate the mutational spectrum of novel somatic mutations, we assessed 41 pairs of tumor-stroma tissues from Chinese patients with CRC, including 29 colon carcinomas and 12 rectal carcinomas. We designed Illumina Custom Amplicon panel to target 43 genes, including genes in the four candidate pathways, as well as several known oncogenes for other cancers. Candidate mutations were validated by Sanger sequencing, and we further used SIFT and PolyPhen-2 to assess potentially functional mutations. We discovered 3 new somatic mutations in gene APC, TCF7L2, and PIK3CA that had never been reported in the COSMIC or NCI-60 databases. Additionally, we confirmed 6 known somatic mutations in gene SMAD4, APC, FBXW7, BRAF and PTEN in Chinese CRC patients. While most were previously reported in CRC, one mutation in PTEN was reported only in malignant endometrium cancer. Our study confirmed the existence of known somatic mutations in the four candidate pathways for CRC in Chinese patients. We also discovered a number of novel somatic mutations in these pathways, which may have implications for the pathogenesis of CRC.     

Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTa_A14Q-dmLT or 3xSTa_N12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STa_A14Q-dmLT and CFA/I/II/IV-STa_N12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa_-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTa_N12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa_-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTa_N12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.