Synthesis of the same two proteins prior to larval diapause and pupation in the spruce budworm,Choristoneura fumiferana

Spruce budworm larvae produce large quantities of two proteins (Choristoneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and CfDAP2) that are diapause related. These proteins appeared soon after hatching and increased in abundance, reaching maximum levels by four days into the 1st instar, and they remained at high levels until three days after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligonucleotide primers designed on the basis of these NH2-terminal sequences were used in RT-PCR to isolate the cDNA fragments coding for these proteins. These PCR fragments were then used as probes to isolate the cDNAs that contained the complete coding region. The 2.5kb mRNAs coding for these proteins started to appear 24hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar larvae until the 2nd instar larvae entered diapause. Low levels of these mRNAs were detected in the 2nd instar larvae that were preparing to enter diapause, in those that were in diapause as well as in those that terminated diapause. Low levels of CfDAP1 mRNA were also detected on days 1 and 2 after ecdysis to the 3rd instar. However, no CfDAP1 and CfDAP2 mRNAs could be detected during the 4th and 5th instar larval stages. The mRNAs reappeared 24hr after the 5th instar larvae molted into the 6th instar and increased to reach maximum levels by 60hr after ecdysis. The mRNA levels remained high until 156hr after ecdysis into the 6th instar (36-48hr before pupal ecdysis), after which they disappeared once again. Immunocytochemical analyses showed that CfDAP1 protein was present in 2nd and 6th instar larval fat body but not in 5th instar larval fat body. Thus, the same two genes were expressed for the first time before C. fumiferana larvae entered diapause and for a 2nd time before pupation.     

The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity ( K _D = 0.47 n M ) and saturable binding with ¹²⁵I-galanin ( B _max = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.     

永久型70kD热休克蛋白在白菜花发育过程中的免疫组织化学定位

利用免疫组织化学技术研究了永久型热休克蛋白ＨＳＣ７０在白菜花各组织中的分布。结果表明：在正常温度条件下,ＨＳＣ７０在小孢子母细胞、四分体细胞、花药壁绒毡层细胞中分布最多,在花原基、花托的维管组织、花粉母细胞以及发育早期的胚珠中的表达也较多。该结果与其他人用核酸杂交、同位素示踪等技术所得结果基本一致,本文对ＨＳＣ７０在白菜花不同组织中的分布与其功能的关系进行了初步讨论。     

天花粉蛋白Y14F/R22L定点突变及其活性研究

利用多聚酶链式反应（ＰＣＲ）技术,对天然天花粉蛋白（ｎＴＣＳ）基因在Ｔｙｒ１４和Ａｒｇ２２两个保守残基处同时进行定点突变,即Ｔｙｒ１４变成Ｐｈｅ,Ａｒｇ２２变成Ｌｅｕ,然后克隆到ｐＥＴ－８ｃ高效表达载体上,构建成重组质粒ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ．经序列分析,定点突变的结果与预先设计的完全一致,突变后的天花粉蛋白命名为Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ．将ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ转化到Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３,ｐＬｙｓＳ）中,进行表达．经ＣＭ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ柱纯化,ＳＤＳ－ＰＡＧＥ鉴定,纯度可达９０％．ＲＩＰ活性测定显示,Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ的活性比ｎＴＣＳ降低了７．５倍,活性变化不显著,因此,ＴＣＳ的Ｔｒｙ１４和Ａｒｇ２２对维持其活性部位构象并不是必需的．但由于Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ在Ｅ．ｃｏｌｉ中的表达量与ｎＴＣＳ相比明显下降,因此,Ｔｙｒ１４和Ａｒｇ２２可能与ＴＣＳ翻译后的折叠有关．     

Proteomic profile of the plant‐pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph

Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control plant‐pathogenic oomycetes such as Phytophthora capsici. The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ technology to determine the target site of the fungicide and potential biomarker candidates of drug efficacy. A total of 1336 unique proteins were identified from the mycelium of wild‐type P. capsici isolate (Hd3) and two pyrimorph‐resistant mutants (R3‐1 and R3‐2) grown in the presence or absence of pyrimorph. Comparative analysis revealed that the three P. capsici isolates Hd3, R3‐1, and R3‐2 produced 163, 77, and 13 unique proteins, respectively, which exhibited altered levels of abundance in response to the pyrimorph treatment. Further investigations, using Cluster of Orthologous Groups of Proteins (COG) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 35 proteins related to the mode of action of pyrimorph against P. capsici and 62 proteins involved in the stress response of P. capsici to pyrimorph. Many of the proteins with altered expression were associated with glucose and energy metabolism. Biochemical analysis using d ‐[U‐¹⁴C]glucose verified the proteomics data, suggesting that the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis. These results also illustrate that proteomics approaches are useful tools for determining the pathways targeted by novel fungicides as well as for evaluating the tolerance of plant pathogens to environmental challenges, such as the presence of fungicides.     

Adding Vitamin E-TPGS to the Formulation of Genexol-PM: Specially Mixed Micelles Improve Drug-Loading Ability and Cytotoxicity against Multidrug-Resistant Tumors Significantly

Genexol-PM, produced by Samyang Company (Korea) is an excellent preparation of paclitaxel (PTX) for clinical cancer treatment. However, it cannot resolve the issue of multidrug resistance (MDR)—a significant problem in the administration of PTX to cancer patients. To increase the efficacy of Genexol-PM against MDR tumors, a mixed micelle capable of serving as a vehicle for PTX was developed, and two substances were chosen as carrier materials: 1) Polyethylene glycol–polylactic acid (PEG-PLA), the original vehicle of Genexol-PM. 2) Vitamin E-TPGS, an inhibitor of P-glycoprotein (P-gp). P-gp has been proven to be the main cause of MDR. In vitro evaluation indicated that the mixed micelle was an ideal PTX delivery system for the treatment of MDR tumors; the mixed micelle also showed a significantly better drug-loading coefficient than Genexol-PM.     

Cloning and Functional Characterization of SAD Genes in Potato

Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.     

Determination of Zygosity in Adult Chinese Twins Using the 450K Methylation Array versus Questionnaire Data

Previous studies have shown that both single nucleotide polymorphisms (SNPs) and questionnaires-based method can be used for twin zygosity determination, but few validation studies have been conducted using Chinese populations. In the current study, we recruited 192 same sex Chinese adult twin pairs to evaluate the validity of using genetic markers-based method and questionnaire-based method in zygosity determination. We considered the relatedness analysis based on more than 0.6 million SNPs genotyping as the golden standards for zygosity determination. After quality control, qualified twins were left for relatedness analysis based on identical by descent calculation. Then those same sex twin pairs were included in the zygosity questionnaire validation analysis. Logistic regression model was applied to assess the discriminant ability of age, sex and the three questions in zygosity determination. Leave one out cross-validation was used as a measurement of internal validation. The results of zygosity determination based on 65 SNPs in 450k methylation array were all consistent with genotyping. Age, gender, questions of appearance confused by strangers and previously perceived zygosity consisted of the most predictable model with a consistency rate of 0.8698, cross validation predictive error of 0.1347. For twin studies with genotyping and\or 450k methylation array, there would be no need to conduct other zygosity testing for the sake of costs consideration.     

Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.     

Real‐time monitoring of incision profile during laser surgery using shock wave detection

Lack of sensory feedback during laser surgery prevents surgeons from discerning the exact location of the incision, which increases duration and complexity of the treatment. In this study we demonstrate a new method for monitoring of laser ablation procedures. Real‐time tracking of the exact three dimensional (3D) lesion profile is accomplished by detection of shock waves emanating from the ablation spot and subsequent reconstruction of the incision location using time‐of‐flight data obtained from multiple acoustic detectors. Here, incisions of up to 9 mm in depth, created by pulsed laser ablation of fresh bovine tissue samples, were successfully monitored in real time. It was further observed that, by utilizing as little as 12 detection elements, the incision profile can be characterized with accuracy below 0.5 mm in all three dimensions and in good agreement with histological examinations. The proposed method holds therefore promise for delivering high precision real‐time feedback during laser surgeries. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)