细胞色素P450介导的果蝇抗药性研究进展

细胞色素P450 (cytochrome P450, CYP450)超基因家族是由一些数量多而功能复杂的血红蛋白酶基因所组成,该代谢酶系作为一种几乎地球上所有需氧生物都存在的重要生存策略,可以调控多种内源物质及外源化合物的代谢,参与了众多重要的生命过程,代谢解毒作用是该酶系重要功能之一。细胞色素P450的代谢解毒作用受药物影响,机体通过改变基因表达量,实现增强代谢解毒,加快机体对于有害物质的代谢,从而使得机体对有害环境产生一定的适应性,进而使得机体产生耐药性或抗药性。本研究说明果蝇细胞色素P450介导的杀虫剂类药物代谢机制及代谢抗性的特点等方面的研究,对明确果蝇的抗药性机制研究具有参考意义。     

Origin of the X1X1X2X2/X1X2Y sex chromosome system of Harttia punctata (Siluriformes,Loricariidae) inferred from chromosome painting and FISH with ribosomal DNA markers

Harttia is a genus in the subfamily Loricariinae that accommodates fishes popularly known as armored catfishes. They show extensive karyotypic diversity regarding interspecific numerical/structural variation of the karyotypes, with the presence of the XX/XY₁Y₂ multiple sex chromosome system, as found in H. carvalhoi. In this context, this study aimed to characterize Harttia punctata chromosomally, for the first time, and to infer the rearrangements that originated the X₁X₁X₂X₂/X₁X₂Y multiple sex chromosome system present in this species. The data obtained in this study, with classical (Giemsa, C-banding and AgNORs) and molecular methodologies (fluorescence in situ hybridization) and chromosome microdissection, indicated that a translocation between distinct acrocentric chromosomes bearing rRNA genes, accompanied by deletions in both chromosomes, might have originated the neo-Y chromosome in this species. The data also suggest that the multiple sex chromosome systems present in H. carvalhoi and H. punctata had an independent origin, evidencing the recurrence of chromosome alterations in species from this genus.     

Dysregulation of Wnt inhibitory factor 1 (Wif1) expression resulted in aberrant Wnt-β-catenin signaling and cell death of the cloaca endoderm,and anorectal malformations

In mammalian urorectal development, the urorectal septum (urs) descends from the ventral body wall to the cloaca membrane (cm) to partition the cloaca into urogenital sinus and rectum. Defective urs growth results in human congenital anorectal malformations (ARMs), and their pathogenic mechanisms are unclear. Recent studies only focused on the importance of urs mesenchyme proliferation, which is induced by endoderm-derived Sonic Hedgehog (Shh). Here, we showed that the programmed cell death of the apical urs and proximal cm endoderm is particularly crucial for the growth of urs during septation. The apoptotic endoderm was closely associated with the tempo-spatial expression of Wnt inhibitory factor 1 (Wif1), which is an inhibitor of Wnt-β-catenin signaling. In Wif1^lacZ/lacZ mutant mice and cultured urorectum with exogenous Wif1, cloaca septation was defective with undescended urs and hypospadias-like phenotypes, and such septation defects were also observed in Shh^−/− mutants and in endodermal β-catenin gain-of-function (GOF) mutants. In addition, Wif1 and Shh were expressed in a complementary manner in the cloaca endoderm, and Wif1 was ectopically expressed in the urs and cm associated with excessive endodermal apoptosis and septation defects in Shh^−/− mutants. Furthermore, apoptotic cells were markedly reduced in the endodermal β-catenin GOF mutant embryos, which counteracted the inhibitory effects of Wif1. Taken altogether, these data suggest that regulated expression of Wif1 is critical for the growth of the urs during cloaca septation. Hence, Wif1 governs cell apoptosis of urs endoderm by repressing β-catenin signal, which may facilitate the protrusion of the underlying proliferating mesenchymal cells towards the cm for cloaca septation. Dysregulation of this endodermal Shh-Wif1-β-catenin signaling axis contributes to ARM pathogenesis.Urorectal development starts at gestational week-4 in humans and at E10.5 in mice, and is divided into three phases: genital tubercle (GT) outgrowth, cloaca septation and urethra tubularization. GT outgrowth is initiated with a paired genital swellings on either side of the cloaca (caudal end of the rectum), which fuse medially to form the future external genitalia. Cloaca septation is the partitioning of the cloaca by urorectal septum (urs) into the rectum and urogenital sinuses (ugs). Urorectal development is indistinguishable in male and female embryos before urethra tubularization. Defective urorectal development results in a number of congenital anomalies frequently accompany with deficient excretory and copulatory functions, greatly influencing the patients'' quality of life. In particular, the anorectal malformations (ARMs) are the most common urorectal defects and affect boys and girls with an incidence of 2–5 in 10 000 live births,^{1, 2} but its pathogenic mechanisms are poorly understood. ARM phenotypes are also partly included in several diseases, such as Currarino syndromes, Townes Brocks Syndromes and VACTERL complex, and these patients display other anomalies including anal fistula, sacral malformations and renal malformations.^{3, 4, 5, 6}During cloaca septation, the urs, an endoderm-lined mesenchyme, elongates from the ventral body wall to the cloaca membrane (cm) (Figure 1a). The proximal cm is regressing when the apical urs endoderm approximates to the cm leading to the formation of the urethral duct (ud) and the anal opening, which signifies the completion of cloaca septation (Figure 1b). After septation, the cm regression associated with the proximal-to-distal urs elongation coupled with fusion of the preputial fold mesoderm along the ventral midline of the tubercle leads to the formation of urethral opening at the distal end of the GT (Figure 1c).Open in a separate windowFigure 1Descent of the urorectal septum and regression of the cloaca membrane during cloaca septation. Schematic diagram of cloaca development showing the descent of urs and regression of proximal cm leads to anal opening after cloaca septation (a–c). Normal cloaca development in rat embryos from E14.5 to E16.5 (a′–c′). Rat treated with ethylenethiourea displayed persistent cloaca at E14.5 (d; n=12), hypospadias-like phenotypes with incomplete cloaca septation at E15.5 (e; n=15) and E16.5 (f); n=9). Scale bar: 50 μmDeletion of Shh, Wnt5a or Bmp7 in mouse embryos resulted in reduced proliferation of the urs mesenchyme, incomplete urs elongation and septation defects.^{7, 8, 9, 10} In addition, the cm was either disintegrated or remained intact in these null embryos, giving rise to hypospadias-like or persistent cloaca phenotypes (Figure 1). Nonetheless, deletion of these genes not only leads to a decrease in cell proliferation but also affect cell apoptosis.^{11, 12}Shh and Wnt signaling are indispensable for the septation process. Shh is expressed in the cloaca endoderm and mediates the proliferation of urs mesenchyme through Gli2. Shh and Gli2 mutants displayed persistent cloaca. However, deletion of Shh eradicated the cm, exposing the unseptated cloaca exteriorly, whereas the cm remained intact in the loss of Gli2.^{7, 13} Wnt5a is crucial for urorectal development and regulates the outgrowth of GT.^{8, 10} Studies suggest that Wnt-β-catenin signaling activity is tightly regulated in urorectal development.^{14, 15} Furthermore, β-catenin expression was downregulated in Shh mutants.¹⁶ Constitutive activated β-catenin in Shh null background can partially rescue defective development of the GT and the cm.^{11, 16} All these indicated that Wnt-β-catenin signaling functions downstream of Shh. How Wnt signaling is regulated during cloaca septation has not been investigated.Wif1 (Wnt inhibitory factor 1) is a secreted Wnt inhibitor, which exerts its inhibitory effect on Wnt signaling by binding directly to Wnt ligands and by preventing the ligands from binding to their cell surface receptors. The presence of Wif1 leads to β-catenin degradation, thereby turning off Wnt-β-catenin signaling.¹⁷ Most of the recent studies focused on the impact of epigenetic silencing of Wif1 in various carcinomas,¹⁸ and restoration of Wif1 activity in cancer cells induced apoptosis of malignant cancers.¹⁹ By contrast, reports on the regulatory functions of Wif1 in embryonic development are limited. Previous study suggested that Wif1 has high affinity to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11;²⁰ and Wif1 regulated chondrogenesis in cartilage-mesenchyme interfaces via the inhibition of Wnt3a-mediated mesenchyme growth in embryos.²¹ A recent study revealed that expression of Wif1 is androgen responsive in prostate bud formation. Loss of Wif1 in prostate glands induced ectopic expression of other secretory Wnt inhibitors to compensate for the loss of Wif1 activity in these mutants.²² Furthermore, Smad1 directly targets the Wif1 promoter and controls Wif1 gene expression in lung epithelial cell development.²³ Taken all these indicated that Wnt-β-catenin signaling is precisely regulated in a number of embryonic developmental processes, and Wif1 modulates the Wnt-β-catenin signaling activity.In this study, we discovered that the finely regulated expression of Wif1 is crucial for cloaca septation, and dysregulated Wif1 expression caused septation defects. Shh and Wif1 were expressed in a complementary manner at the cloaca endoderm, and deletion of Shh induced ectopic Wif1 expression, associated with excessive endoderm apoptosis and septation defects. Comparable septation defects were observed in Wif1^lacZ/lacZ mutant mice, in cultured urorectum with exogenous Wif1, in Shh^−/− mutants and in endodermal β-catenin GOF mutants. In conclusion, this study suggests that Wif1 regulates endodermal cell apoptosis by mediating and regulating Shh-Wnt-β-catenin signaling, thus having an essential role during urorectal development.     

Identification of Mutations in the PYRIN-Containing NLR Genes (NLRP) in Head and Neck Squamous Cell Carcinoma

Head and Neck Squamous Cell Carcinoma (HNSCC) encompasses malignancies that arise in the mucosa of the upper aerodigestive tract. Recent high throughput DNA sequencing revealed HNSCC genes mutations that contribute to several cancer cell characteristics, including dysregulation of cell proliferation and death, intracellular proinflammatory signaling, and autophagy. The PYRIN-domain containing NLR (Nucleotide-binding domain, Leucine rich Repeats – containing) proteins have recently emerged as pivotal modulators of cell death, autophagy, inflammation, and metabolism. Their close physiologic association with cancer development prompted us to determine whether mutations within the NLRP (PYRIN-containing NLR) gene family were associated with HNSCC genome instability and their clinicopathologic correlations. Catastrophic mutational events underlie cancer cell genome instability and mark a point-of-no-return in cancer cell development and generation of heterogeneity. The mutation profiles of 62 patients with primary conventional type HNSCC excluding other histologic variants were analyzed. Associations were tested using Fisher''s Exact test or Mann-Whitney U test. Mutations in NLRP were associated with elevated genome instability as characterized by higher mutation rates. Clinically, NLRP mutations were more frequently found in HNSCC arising in the floor of mouth (50.0%) in comparison with HNSCC at other head and neck locations (14.8%). These mutations were clustered at the leucine rich repeats region of NLRP proteins, and affected NLRP genes were mostly localized at chromosomes 11p15.4 and 19q13.42-19q13.43. Twenty novel NLRP mutations were identified in HNSCC, and mutations in this group of genes were correlated with increased cancer cell genome mutation rates, and such features could be a potential molecular biomarker of HNSCC genome instability.     

温度对星豹蛛实验种群增长的影响

根据星豹蛛(Pardosa astribera Koch)在20℃、23℃、29℃、32℃和35℃6种恒温条件下的发育历期、幼蛛存活率、成蛛产卵率、产卵量和孵化率组建了星豹蛛在不同温度条件下的实验种群增长表,结果表明星豹蛛在所试验的6种恒温条件下其种群数量可保持上升趋势,其中以在29℃条件下种数量增长最快、繁殖1代种群数量对增加37.78倍;以在20℃的条件下种群数量增长最慢,繁殖1代其中种群数量可增加15.77倍。     

Adiponectin Mediated MHC Class II Mismatched Cardiac Graft Rejection in Mice Is IL-4 Dependent

Background

Adiponectin regulates glucose and fatty-acid metabolism but its role in chronic graft rejection mediated by Th2 cytokines remains ill-defined.

Methodology/Principal Findings

Wild type and adiponectin-null mice were used as graft recipients in mouse MHC class II disparate cardiac transplantation (bm12 toB6) and the graft rejection was monitored. In adiponectin-null mice we observed that the cellular infiltrate of eosinophils, CD4⁺ and CD8⁺ T cells was reduced in grafts compared to the controls as was collagen deposition and vessel occlusion. A similar outcome was observed for skin transplants except that neutrophil infiltration was increased. Low levels of IL-4 were detected in the grafts and serum. The effect of adiponectin signaling on IL-4 expression was further investigated. Treatment with AMPK and p38 MAPK inhibitors blocked adiponectin enhanced T cell proliferation in mixed lymphocyte reactions. Inhibition of AMPK reduced eosinophil infiltration in skin grafts in wild type recipients and in contrast AMPK activation increased eosinophils in adiponectin-null recipients. The addition of adiponectin increased IL-4 production by the T cell line EL4 with augmented nuclear GATA-3 and phospho-STAT6 expression which were suppressed by knockdown of adiponectin receptor 1 and 2.

Conclusions

Our results demonstrate a direct effect of adiponectin on IL-4 expression which contributes to Th2 cytokine mediated rejection in mouse MHC class II histoincompatible transplants. These results add to our understanding of the interrelationship of metabolism and immune regulation and raise the possibility that AMPK inhibitors may be beneficial in selected types of rejection.     

Propidium iodide for making heterochromatin more evident in the C-banding technique

Abstract The detection of regions of heterochromatin has been the subject of intense investigation. We investigated an adaptation of the commonly used technique by replacing the nonfluorescent dye, Giemsa, by a fluorescent one, propidium iodide. This adaptation produces greater contrast of the heterochromatic bands in metaphase chromosomes and can be especially valuable when the organisms studied possess heterochromatin that is pale and difficult to visualize. We discuss the interactions of these two dyes with DNA and the excitation of the fluorescent dye when irradiated with ultraviolet light.     

Quick identification of kuraridin, a noncytotoxic anti-MRSA (methicillin-resistant Staphylococcus aureus) agent from Sophora flavescens using high-speed counter-current chromatography

Bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all currently used antibacterial agents and that manifests in all fields of their application. To find more antibacterial agents from natural resources is all the time considered as an important strategy. Sophora flavescens is a popularly used antibacterial herb in Chinese Medicine, from which prenylated flavones were reported as the antibacterial ingredients but with a major concern of toxicity. In our screening on the antibacterial activities of various chemicals of this herb, 18 fractions were obtained from 8 g of 50% ethanol extract on a preparative high-speed counter-current chromatography (HSCCC, 1000 ml). The system of n-hexane/ethyl acetate/methanol/water (1:1:1:1) was used as the two-phase separation solvent. A chalcone named kuraridin was isolated from the best anti-MRSA fraction, together with sophoraflavanone G, a known active ingredient of S. flavescens. Their structures were elucidated by analysis of the NMR spectra. Both compounds exhibited significant anti-MRSA effects, compared to baicalein that is a well known anti-MRSA natural product. More important, kuraridin showed no toxicity on human peripheral blood mononuclear cells (PBMC) at the concentration up to 64 μg/ml while sophoraflavanone G inhibited over 50% of cellular activity at 4 μg/ml or higher concentration. These data suggested that opening of ring A of the prenylated flavones might decrease the toxicity and remain the anti-MRSA effect, from a viewpoint of structure-activity relationship.