Changes in the Ribonuclease Activity of Flax Cotyledons following Inoculation with Flax Rust

There was a significant increase in the ribonuclease activity of both resistant (Bombay) and susceptible (Bison) varieties of flax (Linum usitatissimum L.) 3 to 4 days after inoculation with flax rust (Melampsora lini [Pers.] Lev., race No. 3). A second and much greater increase in the activity of this enzyme occurred only in the susceptible host at later stages of disease development. While a similar increase in ribonuclease level was also caused by mechanical injury, evidence is presented showing qualitative differences between the enzyme from parasitized tissue and that from the mechanically injured cotyledons. Comparison of the enzyme from healthy and inoculated cotyledons and from flax rust revealed the presence of a relatively unstable component and some unique catalytic properties in the enzyme from inoculated cotyledons.     

Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process.     

Basolateral Internalization of GPI-anchored Proteins Occurs via a Clathrin-independent Flotillin-dependent Pathway in Polarized Hepatic Cells

In polarized hepatocytes, the predominant route for apical resident proteins to reach the apical bile canalicular membrane is transcytosis. Apical proteins are first sorted to the basolateral membrane from which they are internalized and transported to the opposite surface. We have noted previously that transmembrane proteins and GPI-anchored proteins reach the apical bile canaliculi at very different rates. Here, we investigated whether these differences may be explained by the use of distinct endocytic mechanisms. We show that endocytosis of both classes of proteins at the basolateral membrane of polarized hepatic cells is dynamin dependent. However, internalization of transmembrane proteins is clathrin mediated, whereas endocytosis of GPI-anchored proteins does not require clathrin. Further analysis of basolateral endocytosis of GPI-anchored proteins showed that caveolin, as well as the small GTPase cdc42 were dispensable. Alternatively, internalized GPI-anchored proteins colocalized with flotillin-2–positive vesicles, and down-expression of flotillin-2 inhibited endocytosis of GPI-anchored proteins. These results show that basolateral endocytosis of GPI-anchored proteins in hepatic cells occurs via a clathrin-independent flotillin-dependent pathway. The use of distinct endocytic pathways may explain, at least in part, the different rates of transcytosis between transmembrane and GPI-anchored proteins.     

Identification of Pathotypes in the Daylily Rust Pathogen Puccinia hemerocallidis

Daylily rust, caused by Puccinia hemerocallidis, has been present in the United States since 2000. In 2003, inoculations with a single isolate of P. hemerocallidis identified daylily cultivars with high levels of resistance to the fungus. The present study was carried out to determine if pathotypes of P. hemerocallidis are present in the south eastern United States. Sixteen isolates of P. hemerocallidis were each inoculated onto leaf segments from 19 daylily cultivars and the resulting disease phenotype assessed. A significant effect of rust isolate on host reaction phenotype was observed for nine of the 19 daylily cultivars. Five of the nine cultivars displayed reaction phenotypes with different isolates of P. hemerocallidis that included at least one susceptible or moderately susceptible and also resistant phenotypes. These results indicate that different pathotypes of the fungus are present in the south east United States. Daylily hybridizers interested in screening for host resistance to P. hemerocallidis will need to include multiple isolates of the fungus to allow for this host specialization.     

Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.

Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate ³⁵S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

     

Investigation of the Internalization of Fluorescently Labeled Lipophilic siRNA into Cultured Tumor Cells

Russian Journal of Bioorganic Chemistry - The attachment of lipophilic molecules of natural origin, which have natural means for cell internalization, to small interfering RNA (siRNA) is an...     

亚麻抗锈病基因M4的特异分子标记

用520个10碱基随机引物对含有亚麻抗锈病基因M4的近等基因系材料NM4及其轮回亲本Bison进行RAPD分析,其中OPA18引物在NM4材料中稳定地扩增出特异的DNA片段。用Bison与NM4杂交产生的F2分离群体进行的遗传连锁性分析表明,RAPD标记OPA18432与M4基因紧密连锁,二者之间的遗传距离为2.1cM。将OPA18432片段回收,克隆和测序,成功地将其转化为SCAR标记。对不同抗源材料的扩增分析表明,该标记是M4基因的特异标记。目前这一标记已成功地应用于亚麻抗锈病基因M4的分子标记辅助选择育种。     

烟草细胞中高效表达的药用蛋白可以提高血清的半衰期

小分子治疗蛋白, 如干扰素α2b(IFNα2)和人生长激素(hGH), 由于其对血清蛋白酶的易感性及分子小, 可被肾迅速清除, 因而一般在血清中的半衰期都很短。化学衍生处理虽可以克服这些问题, 但却要以降低蛋白的生物活性为代价。本研究建立了一种新方法, 使干扰素α2b (IFNα2)和人类生长激素(hGH)在烟草细胞中高效表达为阿拉伯半乳糖聚糖糖蛋白嵌合体(AGP)。这样不仅提高了药用蛋白的产量, 而且提高了其血清半衰期。这种嵌合糖蛋白的分泌量比没有糖基化时高500~1800 倍。重要的是, 与未糖基化的干扰素和人生长激素相比, 这种嵌合糖蛋白的体内血半衰期提高13倍, 生物活性仍保持与未糖基化时相似。这种嵌合糖蛋白注射到小鼠体内未引起免疫性反应。     

Nerve Regeneration and Cholesterol Reutilization Occur in the Absence of Apolipoproteins E and A-I in Mice

Abstract: Apolipoproteins have been implicated in the salvage and reutilization of myelin cholesterol during Wallerian degeneration and the subsequent nerve regeneration. Current evidence suggests that myelin cholesterol complexes with apolipoproteins E and A-I to form lipoproteins that are taken up via low-density lipoprotein receptors on myelinating Schwann cells. We recently reported, however, that apolipoprotein E is not required for nerve regeneration or reutilization of myelin cholesterol. We have now investigated nerve regeneration and the reutilization of cholesterol in mutant mice deficient in both apolipoproteins E and A-I. Morphologic examination of nerves 4 and 12 weeks after crush injury revealed that regeneration proceeded at a normal rate in the absence of these apolipoproteins. Autoradiography of regenerating nerves indicated that prelabeled myelin lipid was reutilized in the regenerating myelin. 3-Hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was down-regulated in the regenerating nerves, indicative of cholesterol uptake via lipoproteins. Prelabeled myelin cholesterol was present in lipoprotein fractions isolated from crushed nerves of mutant mice. These data suggest that there is considerable redundancy in the process of cholesterol reutilization within nerve, and that apolipoproteins other than apolipoproteins E and A-I may be involved in the recycling of myelin cholesterol.     

铁皮石斛锈病病原鉴定及药剂筛选

为了明确铁皮石斛锈病的病原菌种类,筛选其有效防治药剂,通过对云南瑞丽铁皮石斛种植基地调查,运用形态学并结合分子生物学方法对病原菌进行鉴定,选用4种杀菌剂进行田间药效试验。结果表明,铁皮石斛锈病由真菌鞘锈菌(Coleosporium sp.)引起,发生期为每年2～11月,鞘锈菌的夏孢子直接侵染铁皮石斛叶片,以冬孢子越冬。10%苯甲·丙环唑悬浮剂450 mL·hm-2与75%肟菌·戊唑醇水分散粒剂300 g·hm-2的防效显著高于其他两种药剂,3次药后防效分别为82.16%和82.88%,这2种药剂可作为防治铁皮石斛锈病的有效药剂。     

Th1-Mediated Immunity against Helicobacter pylori Can Compensate for Lack of Th17 Cells and Can Protect Mice in the Absence of Immunization

Helicobacter pylori (H. pylori) infection can be significantly reduced by immunization in mice. Th17 cells play an essential role in the protective immune response. Th1 immunity has also been demonstrated to play a role in the protective immune response and can compensate in the absence of IL-17. To further address the potential of Th1 immunity, we investigated the efficacy of immunization in mice deficient in IL-23p19, a cytokine that promotes Th17 cell development. We also examined the course of Helicobacter infection in unimmunized mice treated with Th1 promoting cytokine IL-12. C57BL/6, IL-12 p35 KO, and IL-23 p19 KO mice were immunized and challenged with H. pylori. Protective immunity was evaluated by CFU determination and QPCR on gastric biopsies. Gastric and splenic IL-17 and IFNγ levels were determined by PCR or by ELISA. Balb/c mice were infected with H. felis and treated with IL-12 therapy and the resulting gastric bacterial load and inflammatory response were assessed by histologic evaluation. Vaccine induced reductions in bacterial load that were comparable to wild type mice were observed in both IL-12 p35 and IL-23 p19 KO mice. In the absence of IL-23 p19, IL-17 levels remained low but IFNγ levels increased significantly in both immunized challenged and unimmunized/challenged mice. Additionally, treatment of H. felis-infected Balb/c mice with IL-12 resulted in increased gastric inflammation and the eradication of bacteria in most mice. These data suggest that Th1 immunity can compensate for the lack of IL-23 mediated Th17 responses, and that protective Th1 immunity can be induced in the absence of immunization through cytokine therapy of the infected host.     

Immunochemical Localization of Pathogenesis-Related Proteins Secreted into the Intercellular Spaces of Salicylate-Treated Tobacco Leaves

In tobacco leaves, pathogenesis-related (PR) 1 proteins areabundantly induced by hypersensitive reaction to the infectionwith tobacco mosaic virus (TMV) and by treatment with salicylicacid, and are secreted into the intercellular spaces. To studythe distribution of PR 1 proteins outside of the cells, theimmunogold technique was used with anti-PR 1 antibody. Whensections of salicylate-treated tobacco leaf were reacted withantibody against PR 1a and then with protein A-gold complex,most of the gold label was localized in the intercellular spacesin the region between cells, and a little label was found inthe cytoplasmic matrix and in small electron-dense granulesinside the cells. When salicylate-treated leaves were incubatedwith polygalacturonase and/or cellulase to liberate protoplastsor single cells from the tissue, most of PR 1 proteins weresolubilized far before complete liberation of single cells,suggesting their localization in free spaces or a region susceptableto maceration, such as the secondary cell wall. (Received May 30, 1988; Accepted June 27, 1988)     

Geranylgeraniol Promotes Entry of UT-2 Cells into the Cell Cycle in the Absence of Mevalonate

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5–10 μMGG-OH. Under these growth conditions [³H]GG-OH is actively incorporated into UT-2 proteins. Prominent [³H]geranylgeranylated polypeptides in the size range (19–27 kDa) of the small GTP-binding proteins are observed by SDS–PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [³H]geranylgeranylated cysteine residues. Even though [³H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 μMdid not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.     

A Meiotic Chromosomal Core Consisting of Cohesin Complex Proteins Recruits DNA Recombination Proteins and Promotes Synapsis in the Absence of an Axial Element in Mammalian Meiotic Cells

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.