利用腺病毒DNA与爪蟾卵提取物在非细胞体系中重构细胞核(简报)

1983年,Lohka和Masui首先报道,用精子染色质与卵提取物一起温育,可以组装成具有典型结构的细胞核。这种核不仅具有合成DNA的能力,而且还能进入M期。同年,     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.     

Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method

The amino acids in methanol-soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [³H]aspartic acid and [¹⁴C]asparagine. The aspartic acid pool turned over rapidly with a half-time of <30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as ～25 ng/hr-oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material. © 1994 Wiley-Liss, Inc.     

硅藻变温发酵生产二十碳五烯酸的研究

本文考察不了同温度（10℃～30℃）对硅藻(Nitzschia laevis)合成二十碳五烯酸(EPA)的影响,对不同温度下的发酵过程进行动力学特性分析。在此基础上,提出了EPA合成的变温培养方法：延滞期及对数期初期温度控制在25℃,从对数期中期开始在20℃条件下进行培养。采用此变温培养进行发酵,EPA的含量和产量分别达到了6.00%和291.60 mg·L-1,较采用单一温度(25℃)发酵的最大值分别提高了24.07%和18.81%。     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .     

Pomphorhynchus laevis (Müller) in the flounder, Platichthys flesus L., in the tidal River Thames: population structure, microhabitat utilization and reproductive status in the field and under conditions of controlled salinity

The infection of R. Thames flounders, Platichthys flesus L., at Fulham by the acanthocephalan parasite Pomphorhynchus laevix (Müller) is described in terms of parasite population structure, life-cycle organization, reproductive biology and host microhabitat utilization. The parasites demonstrated 100% prevalence in this tidal but essentially freshwater locality (intestinal intensity 34.47) and were overdispersed in the flounder population (variance/mean ratio = 10.39, k= 1.04). Overall about 11% of the worms occurred in peritoneal cavity sites, the remainder being firmly attached to the gut wall in the posterior region of the intestine and rectum. The P. laevis populations were reproductively active, demonstrating that the flounder acts as a significant final host in this locality. Viable larval stages were identified in Gammarus zaddachi Sexton, a predominantly estuarine amphipod, indicating that P. laevis is likely to be able to complete its life cycle at Fulham. Over half the female parasites examined were gravid and many of the non-gravid worms were inseminated but had not yet started egg production. The proportion of females gravid, the number of ovarian balls and the number of eggs in gravid females was seen to increase with worm size. Both the identity of the intermediate host and the reproductive status of P. laevis in the Thames flounders differ from the Pomphorhynchus/flounder system studied by Kennedy (1984) in the R. Avon, suggesting that the two P. laevis populations may belong at least to discrete subspecies or strains. Parasites in flounders maintained under laboratory conditions in fresh water, 50% sea water and 100% sea water showed similar population, microhabitat and reproductive characteristics to those observed in the field. This suggests that increased salinity has a negligible effect on established parasites in the short term, and therefore that salinity may not form a barrier to the survival and dispersal of the R. Thames parasite when the flounders return to sea.     

Multiple roles of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase cascade in Xenopus laevis

Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine protein kinase that is rapidly activated in response to various growth factors and tumor promoters in mammalian cultured cells. The kinase cascade including MAPK and its direct activator, MAPK kinase (MAPKK), is now believed to transmit various extracellular signals into their intracellular targets in eukaryotic cells. It has been reported that activation of MAPKK and MAPK occurs during the meiotic maturation of oocytes in several species, including Xenopus laevis . Studies with neutralizing antibodies against MAPKK, MAPK phosphatases and constitutively active MAPKK or MAPK have revealed a crucial role of the MAPKK/MAPK cascade in a number of developmental processes in Xenopus oocytes and embryos.     

The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase

Complex cellular processes are driven by the regulated assembly and disassembly of large multiprotein complexes. While we are beginning to understand the molecular mechanism for assembly of the eukaryotic DNA replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Recently, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases have now been shown to ubiquitylate the helicase under different conditions: Cul2^Lrr1 and TRAIP. Here, using Xenopus laevis egg extract cell-free system and biochemical approaches, we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly during S-phase. We show only Ubxn7, however, facilitates efficient replisome disassembly. Ubxn7 delivers this role through its interaction via independent domains with both Cul2^Lrr1 and p97 to allow coupling between Mcm7 ubiquitylation and its removal from chromatin. Our data therefore characterize Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates and a rationale for the efficacy of the Cul2^Lrr1 replisome unloading pathway in unperturbed S-phase.     

Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis

Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as 'non-self'. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose-dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult-type immune system.