Benzo(alpha)pyrene-induced up-regulation of CYP1A2 gene expression: role of adrenoceptor-linked signaling pathways

CYP1A2, a principal catalyst for metabolism of various therapeutic drugs and carcinogens, among others, is in part regulated by the stress response. This study was designed to assess whether catecholamines and in particular adrenergic receptor-dependent pathways, modulate benzo(alpha)pyrene (B(alpha)P)-induced hepatic CYP1A2. To distinguish between the role of central and peripheral catecholamines in the regulation of CYP1A2 induction, the effect of central and peripheral catecholamine depletion using reserpine was compared to that of peripheral catecholamine depletion using guanethidine. The effects of peripheral adrenaline and L-DOPA administration were also assessed. The results suggest that alterations in central catecholamines modulate 7-methoxyresorufin O-demethylase activity (MROD), CYP1A2 mRNA and protein levels in the B(alpha)P-induced state. In particular, central catecholamine depletion, dexmedetomidine-induced inhibition of noradrenaline release and blockade of alpha(1)-adrenoceptors with prazosin, up-regulated CYP1A2 expression. Phenylephrine and dexmedetomidine-induced up-regulation may be mediated, in part, via peripheral alpha(1)- and alpha(2)-adrenoceptors, respectively. On the other hand, the L-DOPA-induced increase in central dopaminergic activity was not followed by any change in the up-regulation of CYP1A2 expression by B(alpha)P. Central noradrenergic systems appeared to counteract up-regulating factors, most likely via alpha(1)- and alpha(2)-adrenoceptors. In contrast, peripheral alpha- and beta-adrenoceptor-related signaling pathways are linked to up-regulating processes. The findings suggest that drugs that bind to adrenoceptors or affect central noradrenergic neurotransmission, as well as factors that challenge the adrenoceptor-linked signaling pathways may deregulate CYP1A2 induction. This, in turn, may result in drug-therapy and drug-toxicity complications.     

Indirect competitive immunoassay for detection of aflatoxin B1 in corn and nut products using the array biosensor

Because of the potential health risks of aflatoxin B₁ (AFB₁), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB₁ in buffer, corn and nut products. AFB₁-spiked foods were extracted with methanol and Cy5-anti-AFB₁ added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB₁. The resulting fluorescence signal decreased as the concentration of AFB₁ in the sample increased. The limit of detection for AFB₁ in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.     

RNA干扰抑制肾小管上皮细胞TSP1表达和TGF-β1活化

血小板反应蛋白1(TSP1)是转化生长因子-β1(TGF-β1)体内重要的活化因子,而后者又是致肾小管间质纤维化的关键因素。观察了针对TSP1的小双链干扰RNA(siRNA-TSP1),抑制由血管紧张素II(AngII)诱导的肾小管上皮细胞TGF-β1过度活化。将根据人TSP1基因序列设计的特异siRNA-TSP1转染人肾小管上皮细胞系(HK-2),利用Western印迹、RT-PCR、流式细胞仪及ELISA等方法,检测了TSP1、TGF-β1及其信号蛋白Smad2与p-Smad2、纤维连接蛋白(FN)和纤溶酶原激活剂抑制物-1(PAI-1)的基因转录水平、蛋白质表达或蛋白质活性。结果显示,siRNA-TSP1能有效转染HK-2细胞,并以剂量依赖方式显著抑制TSP1的基因转录与合成;其对TGF-β1的合成影响较小,但能明显抑制TGF-β1的活化。此外siRNA-TSP1可阻抑TGF-β1依赖的Smad2磷酸化,减少细胞外基质FN以及PAI-1的合成。研究结果提示,由于TSP1是TGF-β1重要的内源性活化因子,故针对TSP1的RNA干扰能在体外有效抑制TSP1表达并相应调抑了TGF-β1的活化。     

Molecular steps of G‐overhang generation at human telomeres and its function in chromosome end protection

Telomeric G‐overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G‐overhang generation at human telomeres is poorly understood. Here, we show that G‐overhangs can undergo cell cycle‐regulated changes independent of telomerase activity. G‐overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C‐strand fill‐in, whereas the sizes of G‐overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C‐strands are precisely defined throughout the cell cycle, indicating that C‐strand resection is strictly regulated. We demonstrate that C‐strand fill‐in is mediated by DNA polymerase α (polα) and controlled by cyclin‐dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G‐overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G‐overhangs and an increase in telomeric DNA damage. Our results suggest that G‐overhang generation at human telomeres is regulated by multiple tightly controlled processes and C‐strand fill‐in is under the control of polα and CDK1.     

GENETIC DATA HINT AT A COMMON DONOR REGION FOR INVASIVE ATLANTIC AND PACIFIC POPULATIONS OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA)1

Gracilaria vermiculophylla (Ohmi) Papenf., an agar‐producing red alga introduced from northeast Asia to Europe and North America, is often highly abundant in invaded areas. To assay its genetic diversity and identify the putative source of invasive populations, we analyzed the mitochondrial cytochrome c oxidase subunit I (cox1) gene from 312 individuals of G. vermiculophylla collected in 37 native and 32 introduced locations. A total of 19 haplotypes were detected: 17 in northeast Asia and three in Europe and eastern and western North America, with only one shared among all regions. The shared haplotype was present in all introduced populations and in ～99% of individuals in the introduced areas. This haplotype was also found at three native locations in east Korea, west Japan, and eastern Russia. Both haplotype and nucleotide diversities were extremely low in Europe and North America compared to northeast Asia. Our study indicates that the East Sea/Sea of Japan is a likely donor region of the invasive populations of G. vermiculophylla in the east and west Atlantic and the east Pacific.     

Gastrophysa polygoni herbivory on Rumex confertus: single leaf VOC induction and dose dependent herbivore attraction/repellence to individual compounds

We report large induction (>65_fold increases) of volatile organic compounds (VOCs) emitted from a single leaf of the invasive weed mossy sorrel, Rumex confertus Willd. (Polygonaceae), by herbivory of the dock leaf beetle, Gastrophysa polygoni L. (Coleoptera: Chrysomelidae). The R. confertus VOC blend induced by G. polygoni herbivory included two green leaf volatiles ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate) and three terpenes (linalool, ß-caryophyllene, (E)-ß-farnesene). Uninjured leaves produced small constitutive amounts of the GLVs and barely detectable amounts of the terpenes. A Y-tube olfactometer bioassay revealed that both sexes of adult G. polygoni were attracted to (Z)-3-hexenal and (Z)-3-hexen-1-yl acetate at a concentration of 300 ng h⁻¹. No significant G. polygoni attraction or repellence was detected for any VOC at other concentrations (60 and 1500 ng h⁻¹). Yet, G. polygoni males and females were significantly repelled by (or avoided) at the highest test concentration (7500 ng h⁻¹) of both GLVs and (E)-ß-farnesene. Mated male and female G. polygoni might be attracted to injured R. confertus leaves, but might avoid R. confertus when VOC concentrations (especially the terpene (E)-ß-farnesene) suggest high overall plant injury from conspecifics, G. viridula, or high infestations of other herbivores that release (E)-ß-farnesene (e.g., aphids). Tests in the future will need to examine G. polygoni responses to VOCs emitted directly from uninjured (constitutive) and injured (induced) R. confertus, and examine whether R. confertus VOC induction concentrations increase with greater tissue removal on a single leaf and/or the number of leaves with feeding injury.     

Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China

Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei ({"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) or S. decipiens ({"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).     

Free radicals impair the anti-oxidative stress activity of DJ-1 through the formation of SDS-resistant dimer

DJ-1 is a causative gene for familial Parkinson’s disease (PD). Loss-of-function of DJ-1 protein is suggested to contribute to the onset of PD, but the causes of DJ-1 dysfunction remain insufficiently elucidated. In this study, we found that the SDS-resistant irreversible dimer of DJ-1 protein was formed in human dopaminergic neuroblastoma SH-SY5Y cells when the cells were exposed to massive superoxide inducers such as paraquat and diquat. The dimer was also formed in vitro by superoxide in PQ redox cycling system and hydroxyl radical produced in Fenton reaction. We, thus, found a novel phenomenon that free radicals directly affect DJ-1 to form SDS-resistant dimers. Moreover, the formation of the SDS-resistant dimer impaired anti-oxidative stress activity of DJ-1 both in cell viability assay and H₂O₂-elimination assay in vitro. Similar SDS-resistant dimers were steadily formed with several mutants of DJ-1 found in familial PD patients. These findings suggest that DJ-1 is impaired due to the formation of SDS-resistant dimer when the protein is directly attacked by free radicals yielded by external and internal stresses and that the DJ-1 impairment is one of the causes of sporadic PD.