A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

Effects of fibrillin-1 degradation on microfibril ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.     

Significantly greater antioxidant anticancer activities of 2,3-dehydrosilybin than silybin

Silybin or silymarin extract has been used to treat liver diseases, and has now been entered into clinical trials for cancer treatment. Here, we compared antioxidant and anticancer activities between silybin and its oxidized form 2,3-dehydrosilybin (DHS). With IC50 at three-fold lower concentrations than silybin, DHS inhibited reactive oxygen species generation in glucose-glucose oxidase system and HepG2 cells. Compared with silybin, DHS elicited greater protection against H2O2-induced HepG2 cell death and galactosamine-induced liver injury in vivo. It is known that oxidants induce releases of metalloproteinases (MMP)-2,-9 which are responsible for invasive and metastasis potentials of transformed cells. DHS at 10 microM markedly inhibited MMP-2,-9 releases as well as invasiveness, while silybin at 90 microM had marginal effects. DHS but not silybin at 30 microM induced apoptosis and loss of mitochondrial membrane potentials. LD50 of DHS was five-fold lower than that of silybin. Our data suggest that DHS may be more useful therapeutically than silybin.     

Immunogold Localisation of the Intermediate Chain within the Protein Complex of Cytoplasmic Dynein

It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain. Both antibodies recognised the intermediate chain by sodium dodecyl sulphate–polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex. The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein. The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain.     

The matrilins--adaptor proteins in the extracellular matrix

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.     

Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares

The treatment of early pregnant mares with a history of repeated early embryonic loss with the progestin altrenogest has become routine; however no controlled studies on the efficiency of altrenogest to prevent embryonic losses are available so far. In the present study, we have investigated effects of altrenogest treatment in mares on conceptus development and the secretion of LH, progesterone, and eCG until day 100 of pregnancy. In addition, differences related to age of mares were assessed. Mares were treated with altrenogest (0.044 mg/kg per os once daily) or sunflower oil (10 ml per os once daily) from day 6 to day 100 after ovulation. Blood samples for analysis of LH, progesterone, and eCG were collected. The size of the embryonic vesicle and embryo/fetus was determined by ultrasound. No difference in the per cycle pregnancy rate between altrenogest-treated (75%) and sunflower oil-treated mares (74%) was detected (n.s.). A significant effect of age but not of altrenogest treatment on mean diameter of the embryonic vesicle was found between days 12 and 22 of pregnancy (e.g. day 15: control, 4-8 years: 22.9 ± 1.0 mm, >8 years: 22.0 ± 1.7 mm, altrenogest, 4-8 years: 26.1 ± 2.0 mm, >8 years: 20.4 ± 1.0 mm, P < 0.05). A significant effect of age and treatment on size of the embryo proper between days 30 and 45 was detected (P < 0.05). In the control group but not in the altrenogest group, size of the embryo proper respective fetus was negatively correlated with age of the mares (day 30: r = −0.834, P < 0.05; day 35: r = −0.506, P < 0.05). Plasma concentrations of LH and progesterone were neither effected by age nor by treatment of mares, but significant effects of age and altrenogest treatment on eCG concentrations between days 40 and 130 were detected (P < 0.05). The present study demonstrates for the first time a positive influence of altrenogest-treatment on a retarded development of the embryo respective fetus around the beginning of placentation in mares older than 8 years.     

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA‐generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain‐derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca²⁺‐dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre‐miR16, was impaired. Decreased production of miR‐16‐5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane‐targeted TRBP. Moreover, miR‐16‐5p or membrane‐targeted TRBP expression blocked BDNF‐induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity‐dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.     

含羞草(Mimosa pudica L.)叶片匀浆悬浮液的滞后环

由于受力后叶子立即发生运动 ,含羞草是一个研究力对于生物细胞作用的良好模型。在以往的研究中 ,人们认为此种现象与受力后渗透压改变、离子通道被激活、细胞骨架的动态变化有关。该文旨在通过观察含羞草叶片和叶柄匀浆悬浮液的应力 切变率滞后环变化 ,揭示含羞草的力学性质。在用于比较的含羞草、叶下珠和猪骨骼肌匀浆悬浮液以及水 4个系统中 ,只有含羞草系统具有明显的逆时针滞后环轨迹 ,而其它的 3个系统均不存在。以上结果提示 ,在含羞草的匀浆悬浮液系统中 ,有一种或多种物质 (可能是蛋白质和细胞骨架 )在剪切应力作用过程中由颗粒状结构向网状结构转变 ,由无序结构向有序结构转变 ,由液体结构向黏弹性状态转变 ,而当力撤除以后再缓慢恢复。     

Cleavage of Zearalenone by Trichosporon mycotoxinivorans to a Novel Nonestrogenic Metabolite

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C₁₈H₂₄O₇, was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H]⁻ and at m/z 375.1416 [M+Na]⁺. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay.Zearalenone (ZON) is the main member of a growing family of biologically important “resorcylic acid lactones” (RALs), which have been found in nature. ZON is produced by several Fusarium species, which colonize maize, barley, oat, wheat, and sorghum and tend to develop ZON during prolonged cool, wet growing and harvest seasons (38). Maize is the most frequently contaminated crop plant, and therefore, ZON can be found frequently in animal feeding stuff. Occurrence, toxicity, and metabolism data of ZON were summarized by the European Food Safety Authority (EFSA) (5) and in recent reviews (12, 38).The potent xenohormone ZON leads to hyperestrogenism symptoms and in extreme cases to infertility problems, especially in pigs (15). Ovarian changes in pigs have been noted with toxin levels as low as of 50 μg/kg in the diet (1). Ruminants are more tolerant to ZON ingestion; however, hyperestrogenic syndrome, including restlessness, diarrhea, infertility, decreased milk yields, and abortion, have been well documented with cattle and sheep (4, 29).Because widespread ZON contamination in feed can occur in problematic years, efficient ways to detoxify are desirable. The transformation of mycotoxins to nontoxic metabolites by pure cultures of microorganisms or by cell-free enzyme preparations (3) is an attractive possibility. Microbial metabolization of ZON to alpha-ZOL and beta-ZOL cannot be regarded as detoxification, because both ZOL products are still estrogenic (14). Also, formation of ZON-glucosides and -diglucosides (8, 17) and ZON-sulfate (7) cannot be considered true detoxification but rather formation of masked mycotoxins, because the conjugates may be hydrolyzed during digestion (11, 23), releasing ZON again (2).As the estrogenic activity of ZON and its derivates can be explained by its chemical structure, which resembles natural estrogens (20), it can be expected that cleavage of the lactone undecyl ring system of ZON results in permanent detoxification.El-Sharkawy and Abul-Hajj (9) were the first to report inactivation of ZON after opening of the lactone ring by Gliocladium roseum. This filamentous fungus was capable of metabolizing ZON in yields of 80 to 90%. Also Takahashi-Ando et al. (31) described the degradation reaction of ZON with Clonostachys rosea (synonym of G. roseum). A hydrolase (encoded by a gene designated ZHD101) cleaves the lactone ring, and as recently proved (37; unpublished data) by subsequent decarboxylation of the intermediate acid, the compound 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1′E-undecene-6′-one is formed. In contrast to ZON and 17β-estradiol, which showed potent estrogenic activity, this cleavage product did not show any estrogenic activity in the human breast cancer MCF-7 cell proliferation assay (16). Further details, e.g., on the conditions of the maximum activity of ZHD101 and its exploitation in genetically modified grains, can be found in later published work of this research group (32, 33).Only a few authors reported the loss of estrogenicity in microbial metabolites of ZON, which are based on reactions other than cleavage of the lactone undecyl ring system. El-Sharkawy and Abul-Hajj demonstrated (10) that binding to rat uterine estrogen receptors requires a free 4-OH phenolic group (devoid of methylation or glycosylation). Loss of estrogenicity was, for instance, observed with 2,4-dimethoxy-ZON, one of the metabolites produced by Cunninghamella bainieri ATCC 9244B. Nevertheless, this rule cannot be generalized, as 8′-hydroxyzearalenone formed by Streptomyces rimosus NRRL 2234, despite having a free 4-phenolic hydroxyl group, did not bind to the estrogen receptor. Also, other authors reported that 8′-hydroxyzearalenone and 8′-epi-hydroxyzearalenone are nonestrogenic (13). However, so far, no practical application in feed or food detoxification has been found for the microorganisms producing these compounds.It has been shown previously that the yeast Trichosporon mycotoxinivorans has a very high capability to degrade both ochratoxin A (OTA) and ZON (22, 26, 27). When T. mycotoxinivorans is used as a feed additive preparation, microbial degradation of the mycotoxins is assumed to take place in the gastrointestinal tract of the animal after consumption of contaminated feed. The protective effect of T. mycotoxinivorans against OTA toxicity has already been shown with broiler chicken (24).In the present study we report the isolation, analytical characterization, and structure elucidation, as well as the evaluation, of the estrogenic activity of the main degradation product of ZON produced by T. mycotoxinivorans.