The intersectional hybrid between Weigela hortensis and W. maximowiczii (Caprifoliaceae)

Morphologically intermediate plants between Weigela hortensis (Siebold & Zucc.) K.Koch and W. maximowiczii (S.Moore) Rehder have been found in Miyagi and Yamagata Pref., northern Japan. Quantitative character analyses of flowers, pollen stainability and molecular analyses indicated that the intermediate plants were hybrids of those two species. This is the first record of an intersectional hybrid with W. maximowiczii (sect. Weigelastrum ) as one of the parent species. The morphological differences among hybrid individuals imply the possibility of backcrosses or formation of second or later generations of hybrids, although those may be quite rare because of a low frequency of viable pollen grains. Causes of hybridization between two distantly-related species in Weigela are discussed. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 369–380.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Existence of Sites for Anions and Divalent Cations in the Solubilized γ-Aminobutyric Acid/Benzodiazepine Receptor Complex

This study evaluated the ability of gamma-aminobutyric acid (GABA), baclofen, monovalent anions, divalent cations, and various combinations thereof to protect solubilized benzodiazepine (BZ) receptors of types 1 and 2, when contained together on the complex, against heat inactivation. Neither anions, cations, nor GABA alone provided significant protection of solubilized BZ receptors against heat, but inclusion of monovalent anions or divalent cations together with 500 microM GABA did afford protection. Monovalent anions combined with GABA (500 microM) provided 50% to full protection. Divalent cations, such as CaCl2 (2.5 mM) or MgCl2 (2.5 mM) in the presence of GABA (500 microM) yielded 45% and 24% protection, respectively. Other divalent cations tested (Zn2+, Hg2+, Co2+, and Ni2+) were poor protectors, even when combined with GABA. Monovalent anions (200 mM NaCl) and divalent cations (5 mM CaCl2) when tested together provided no protection. Similarly, baclofen (the GABA-B agonist) provided no protection, either alone or together with anions or divalent cations. These results indicate that the independent but interacting recognition sites of GABA, BZ, anions, and divalent cations, previously detected in the membrane-bound state, are retained in the solubilized state.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Regional Alterations in Rat Brain Neurotransmitter Systems Following Chronic Lithium Treatment

Abstract: Chronic, but not acute, consumption of lithium leads to a significant decrease in serotonin and GABA receptor binding in selected regions of the rat brain, with no changes noted in P-adrenergic or cholinergic muscarinic receptor binding. In addition, the concentration of β-methoxytyramine, a dopamine metabolite, in the corpus striatum was increased in the animals treated chronically with lithium, suggesting a possible enhancement in dopamine release, or inhibition of uptake, in this brain area. In contrast, chronic consumption of rubidium had no effect on any of the parameters studied. The results suggest that lithium administration causes selective changes in brain neurotransmitter receptor systems and that the net result of these changes may be a decrease in GABAergic and serotoninergic activity. The fact that these alterktions are noted only after chronic administration suggests that they may be related to the therapeutic action of lithium in the prophylactic treatment of recurrent manic- depressive psychosis.     

Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Competitive and facilitative relationships among three shrub species,and the role of browsing intensity and rooting depth in the Succulent Karoo,South Africa

Abstract. The nearest‐neighbour technique is used to infer competition and facilitation between the three most abundant species in a semi‐arid region of western South Africa. Relationships among the shrubs Leipoldtia schultzei and Ruschia robusta, which are leaf‐succulent members of the Mesembryanthemaceae (‘mesembs’) and Hirpicium alienatum a non‐succulent Asteraceae, were compared on two adjacent sites with different histories of browsing intensity. Competition was more prevalent and more important than facilitation. The only evidence for facilitation was found at the heavily‐browsed site where the palatable Hirpicium was larger under the unpalatable Leipoldtia. Generally the prevalence and importance of competition was reduced at the heavily‐browsed site. Strong evidence was obtained for intraspecific competition in each of the three species; also, competition was evident between the two mesembs, where Leipoldtia was competitively dominant over Ruschia, although neither species inhibited Hirpicium. Minimal competition between the mesembs and the asteraceous shrub was interpreted in terms of differentiation in rooting depth, and competition within the mesembs, in terms of overlap in rooting depth. The mesembs had the bulk of their roots in the top 5 cm of soil, while the asteraceous shrub had the bulk of its roots, and all its fine roots, at greater depths. The shallow‐rooted morphology of the mesembs is well adapted to utilize small rainfall events, which occur frequently in the Succulent Karoo, and do not penetrate the soil deeply. Modifications of existing methods are applied for analysing nearest‐neighbour interactions.     

表皮生长因子受体基因突变检测方法改进及初步临床验证

目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。