Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

GC32细胞对三种虫媒病毒的敏感性研究

用胃癌细胞株ＧＣ３２,对乙型脑炎、基孔肯雅、兰加特病毒进行敏感性研究。并用ＢＨＫ２１细胞作对照,发现ＧＣ３２细胞对两种病毒的敏感性与对照细胞ＢＨＫ２１极为接近。ＢＨＫ２１和ＧＣ３２细胞对基孔肯雅、乙型脑炎和兰加特的免疫荧光实验,于感染后４８ｈ或７２ｈ都出现＋＋＋～＋＋＋＋的阳性结果。两种细胞对基孔肯雅和乙型脑炎病毒都形成空斑,ＢＨＫ２１细胞对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为５．６５和５．３６;ＧＣ３２对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为６．４８和５．６１。实验表明,ＧＣ３２细胞可以作为有关病毒实验的理想细胞株。     

Newcastle disease virus (NDV) induces protein oxidation and nitration in brain and liver of chicken: Ameliorative effect of vitamin E

The present study was aimed at investigating the therapeutic efficacy of vitamin E on oxidative injury in brain and liver of Newcastle disease virus (NDV) challenged chickens. We have analyzed the xanthine oxidase (XOD) activity; uric acid (UA) levels and superoxide radical generation by using electron spin resonance spectroscopy. Further, protein oxidation, nitration and apoptosis were evaluated in the brain and liver of the control, NDV-infected and NDV + Vit. E treated groups. A significant elevation was observed in XOD activity and UA levels in brain (p < 0.001) and liver (p < 0.05) of NDV infected birds when compared to controls. Further, significant increase in the production of superoxides, enhanced intracellular protein carbonyls and nitrates were observed in the brain and liver of NDV-infected birds over healthy subjects. Apoptosis studies also suggested that a larger number of TUNEL positive cells were observed in brain and a moderately in liver of NDV-infected chickens. However, all these perturbations were significantly ameliorated in NDV + Vit. E treated chickens as compared to NDV-infected birds. Taken together, our results suggested that NDV-induced neuronal and hepatic damage at least in part mediates oxidative stress and on the other hand, supplementation of vitamin E mitigates NDV-induced oxidative damage thereby protects brain and liver of chickens. These findings could provide new insights into the understanding of NDV pathogenesis and therapeutic effects of dietary antioxidants.     

Synthesis, crystal structures and, antibacterial and antiproliferative activities in vitro of palladium(II) complexes of triphenylphosphine and thioamides

Palladium(II) complexes with triphenylphosphine (PPh₃) and thioamides of the general formulae, [Pd(L)₂(PPh₃)₂]Cl₂ and [Pd(L)₂(PPh₃)₂] have been prepared and characterized by elemental analysis, IR and NMR (¹H, ¹³C and ³¹P) methods, and two of them (trans-[Pd(PPh₃)₂(Dmtu)₂]Cl₂·(H₂O)(CH₃OH)_0.5 (1) and trans-[Pd(PPh₃)₂(Mpy)₂] (2)) by X-ray crystallography; where L = thiourea (Tu), methylthiourea (Metu), N,N′-dimethylthiourea (Dmtu), tetramethylthiourea (Tmtu), 2-mercaptopyridine (Mpy), 2-mercaptopyrimidine (Mpm) and thionicotinamide (Tna). The spectral data of the complexes are consistent with the sulfur coordination of thioamides to palladium(II). The crystal structures of the complexes show that (1) has ionic character consisting of [Pd(PPh₃)₂(Dmtu)₂]⁺² cations and uncoordinated Cl⁻ ions, while (2) is a neutral complex with Mpy behaving as anionic thiolate ligand. The coordination environment around palladium in (2) is nearly regular square-planar, while in (1) the trans angles show significant distortions from 180°. The complexes were screened for antibacterial effects, brine shrimps lethality bioassay and antitumor activity. These complexes showed significant activities in most of the cases against the tested bacteria as compared to that of a standard drug. Their antitumor activity against prostate cancer cells (PC3) is comparable with doxorubicin, together with no cytotoxic effects in brine shrimps lethality bioassay study.     

Potential role of cyanidin 3-glucoside (C3G) in diabetic cardiomyopathy in diabetic rats: An in vivo approach

The present study aimed to evaluate the importance of cyanidin 3-glucoside (C3G) of diabetic cardiomyopathy in diabetic rats. The rats were induced with diabetic using streptozotocin and total triglyceride (TG) and total cholesterol (TC) were determined. The range of myocardial enzymes such as aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LD) were also estimated, further, the Immuno histochemical analysis and western blot investigation were determined for the actual activity of C3G. Results indicated that the marker enzymes such as CK, LD and AST were significantly (P < 0.05) increased in STZ administered rats (DM group), while the levels of these elevated marker enzymes of cardiac injury significantly (P < 0.05) declined in the DM + C3G group, as compared to the diabetic group of rats. Additionally, a decrease in the level of TNF-alpha and interleukin-6, was noticed in the C3G treated group as compared to diabetic group. Finally, blotting analysis clearly confirmed that theC3G treatment resulted to higher level response of Bcl-2 and lower level response of caspase-3 and BAX. In conclusion, C3G a natural antioxidant may prevent cardiovascular complications by ameliorating oxidative damage, inflammation, metabolic dysfunctions and apoptosis pathways in type 2 diabetes.     

半抗原特异性抗体的筛选及亲和力成熟

噬菌体抗体库技术的出现开创了一条便捷的基因工程抗体生产路线,为小分子半抗原抗体的制备提供了一条新途径,具有极大的应用潜力,但得到的抗体片段的亲和力普遍较全分子差,需要优化筛选策略及抗体的体外亲和力成熟来解决这一问题。     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Analysis of odorant-binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, which produces lepidopteran Type II sex pheromone components

Information on the olfactory system in antennae of Geometridae moths is very limited, and odorant-binding proteins (OBPs) working as transporters of lipophilic odors have not been identified. In the first investigation on this family of insects, we examined antennal OBPs of the Japanese giant looper, Ascotis selenaria cretacea. RT-PCR experiments using several pairs of degenerate primers designed from known cDNA sequences encoding lepidopteran OBPs successfully amplified partial sequences of two pheromone-binding proteins (PBPs), named AscrPBP1 and AscrPBP2 in reference to their corresponding nucleotide sequence homologies with other PBPs. Using 5′- and 3′-rapid amplification of cDNA end strategies, a cDNA clone for AscrPBP1 encoding a protein of 141 amino acids was isolated. Western blotting with the antiserum against recombinant AscrPBP1 overexpressed in Escherichia coli showed that the AscrPBP1 gene was more strongly expressed in male antennae than in female antennae. Furthermore, natural AscrPBP1was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C₁₉ chain. The result indicated that AscrPBP1 bound not only the pheromone components, 3,6,9-nonadecatriene and its 3,4-epoxy derivative, but also unnatural 6,7- and 9,10-epoxy derivatives. While no general odorant-binding proteins (GOBPs) were amplified in the RT-PCR experiments, two antisera prepared from GOBP1 and GOBP2 of Bombyx mori suggested the occurrence of at least two GOBPs in the A. s. cretacea antennae.     

CCK—8、M—ENK免疫反应结构在猫延髓吻侧腹侧区的分布

用免疫组化 ABC 技术,观察了八肽缩胆囊素(CCK—8),甲硫氨酸脑啡肽(M—ENK)免疫反应(IR)结构在猫延髓吻侧腹侧区的分布。结果表明:CCK—8—IR 细胞分内、外两群:内侧群细胞分布于巨细胞网状核(NGc)、旁巨细胞外侧核(PGL)以及下橄榄核背外侧的网状结构,从吻侧向尾侧逐渐减少;外侧群细胞分布于外侧网状核(LRN)及其背内侧网状结构,从吻侧向尾侧逐渐增多。在中缝苍白核(Rpa)、中缝大核(Rm)仅见少量 CCK—8—IR 细胞。察见 CCK—8—IR纤维主要有3种:粗、细和终末前纤维。CCK—8—IR 纤维在面后核、疑核以及二核紧邻的网状结构最为密集;在 PGL 密度中等;在 NGc、LRN、Rpa 和 Rm 稀疏分布。M—ENK—IR 细胞和纤维分布于 Rpa、Rm、NGc、PGL 和 LRN,此外在面后核、疑核以及二核紧邻的网状结构可见较密集的纤维。     

新生大鼠窦房结组织和培养细胞的心钠素(ANF)表达的定量形态学观察

本实验采用窦房结细胞纯化培养、免疫细胞化学、免疫组化、免疫电镜 （ｐＡｇ 技术） 和图像分析等多种方法,研究了新生ＳＤ大鼠窦房结的ＡＮＦ表达。结果提示： 窦房结原位组织和培养细胞的胞浆内均可见ＡＮＦ阳性反应颗粒。培养细胞的核附近电子致密颗粒有１０ｎｍ ＡＮＦ免疫反应的ｐＡｇ 颗粒。窦房结ＡＮＦ的含量和活性明显低于心房 （Ｐ＜ ００１）。本文还对新生鼠窦房结ＡＮＦ的生理作用进行了讨论