Characterization of metamorphic changes in anuran larval epidermis using lectins as probes

The skin of an adult frog of Xenopus laevis was characterized by the reactivity of 20 lectins. The lectins were classified into six groups in their binding to the epidermal cells: Lycopersicon esculentum lectin (LEL)-type which was positive for all epidermal cells; Pisum sativum agglutinin (PSA)-type for stratum germinativum; succinylated wheat germ agglutinin (sWGA)-type for strata spinosum, granulosum and corneum; Dolichos biflorus agglutinin (DBA)-type for strata germinativum and spinosum; peanut agglutinin (PNA)-type for stratum spinosum; and Ulex europaeus agglutinin (UEA-I)-type for strata granulosum and corneum. PSA and sWGA were utilized as markers of mitotically active germinative cells and the differentiated cells of the epidermis, respectively, to describe the metamorphic conversion of larval epidermal cells to adult type. PSA stained all epidermal cells of tadpoles before metamorphic climax. At the end of metamorphosis, PSA-positive cells were restricted to cells in the basal layer of body epidermis while all the tail epidermis remained PSA-positive. The other cell marker, sWGA, only stained apical cells in tadpole epidermis. During the metamorphic climax, sWGA-positive cells appeared in the cells beneath the stratum corneum of the body region, but not in the tail region. The present study demonstrates that PSA and sWGA are useful to investigate metamorphic changes in tadpole epidermal cells.     

Isolation and characterization of the nuclear matrix from the male xenopus laevis following estrogen administration: Kinetics of [3H] uridine incorporation

At various times following estorgen administration, the nuclear matrix was isolated from the liver of male Xenopus laevis by sucrose gradient centrifugation of nuclei treated with a high-salt buffer and DNase I in the presence of a proteolytic inhibitor (PMSC - phenylmethyl sulfonyl chloride). Electron micrographs of the nuclear matrix demonstrate a sponge-like network attached to a well-defined inner envelope with a ribosome-free outer envelope. Chemical analyses show that the HSB-DNase-treated nuclei consist of 16% DNA, 2% RNA, and 82% protein, a composition that is consistent with that of nuclear matrices isolated from other species. The specific activity of the matrix-associated RNA following estrogen treatment appears to be maximally enhanced after 5 h and decreases until approximately 12 h, when the activity begins to increase again.     

Protein pattern of Xenopus laevis embryos grown in simulated microgravity

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.     

Over 4100 protein identifications from a Xenopus laevis fertilized egg digest using reversed‐phase chromatographic prefractionation followed by capillary zone electrophoresis–electrospray ionization–tandem mass spectrometry analysis

A tryptic digest generated from Xenopus laevis fertilized embryos was fractionated by RPLC. One set of 30 fractions was analyzed by 100‐min CZE‐ESI‐MS/MS separations (50 h total instrument time), and a second set of 15 fractions was analyzed by 3‐h UPLC‐ESI‐MS/MS separations (45 h total instrument time). CZE‐MS/MS produced 70% as many protein IDs (4134 versus 5787) and 60% as many peptide IDs (22 535 versus 36 848) as UPLC‐MS/MS with similar instrument time (50 h versus 45 h) but with 50 times smaller total consumed sample amount (1.5 μg versus 75 μg). Surprisingly, CZE generated peaks that were 25% more intense than UPLC for peptides that were identified by both techniques, despite the 50‐fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically pumped nanospray interface used in CZE. This report is the first comparison of CZE‐MS/MS and UPLC‐MS/MS for large‐scale eukaryotic proteomic analysis. The numbers of protein and peptide identifications produced by CZE‐ESI‐MS/MS approach those produced by UPLC‐MS/MS, but with nearly two orders of magnitude lower sample amounts.     

南方红豆杉丛枝菌根(AM)的研究

研究了南方红豆杉根部丛枝菌根真菌(AMF)侵染情况、菌根形态结构以及根际土中AMF孢子的种类与数量.结果显示:南方红豆杉可与AMF形成典型的丛枝-泡囊型菌根,侵染率在71.2%~94.4%,但是历山、蟒河自然保护区的侵染强度优于人工栽培区;在南方红豆杉根际土中共分离鉴定出5种AMF,无梗囊霉属1种、球囊霉属4种,分别是:光壁无梗囊霉、地表球囊霉、地球囊霉、缩球囊霉、明球囊霉,其中光壁无梗囊霉为优势种;南方红豆杉的根由表皮、外皮层、内皮层、中柱组成,AMF只侵染表皮层、内皮层,不能侵染中柱.这为将来利用AMF接种技术进行南方红豆杉的繁殖、移植栽培以及紫杉醇的积累等研究提供了理论依据.     

爪蟾(Xenopus laevis)的雄性二倍体成体的产生

野生型爪蟾的色素基因是显性,a~p白化型的色素基因是隐性。用a~p型精子与野生型卵子受精,以UV照射卵子使卵核失活,再用静液压方法抑制受精卵的第一次核分裂,结果产生雄核发育的二倍体(androgenetic diploid)。它们是核质杂种。这些杂种发育时黑色素细胞出现时期同野生型一致。色素细胞的数目和颜色介于野生型和白化型之间。当白化型蝌蚪本来就极少的色素细胞消退时,杂种蝌蚪仍有许多色素细胞,直至变态完成两个月后色素才完全消退。因此核质杂种黑色素细胞的发育并维持到变态后,归因于野生型卵细胞质对a~p型核的作用。??实验还证明了仅具有父本基因组的爪蟾能发育至成体,性成熟,均为雄性。其精子与正常卵结合产生发育正常的下一代。现已变态成为幼蟾。     

Recovery of the Xenopus laevis heart from ROS-induced stress utilizes conserved pathways of cardiac regeneration

Urodele amphibians and some fish are capable of regenerating up to a quarter of their heart tissue after cardiac injury. While many anuran amphibians like Xenopus laevis are not capable of such feats, they are able to repair lesser levels of cardiac damage, such as that caused by oxidative stress, to a far greater degree than mammals. Using an optogenetic stress induction model that utilizes the protein KillerRed, we have investigated the extent to which mechanisms of cardiac regeneration are conserved during the restoration of normal heart morphology post oxidative stress in X. laevis tadpoles. We focused particularly on the processes of cardiomyocyte proliferation and dedifferentiation, as well as the pathways that facilitate the regulation of these processes. The cardiac response to KillerRed-induced injury in X. laevis tadpole hearts consists of a phase dominated by indicators of cardiac stress, followed by a repair-like phase with characteristics similar to mechanisms of cardiac regeneration in urodeles and fish. In the latter phase, we found markers associated with partial dedifferentiation and cardiomyocyte proliferation in the injured tadpole heart, which, unlike in regenerating hearts, are not dependent on Notch or retinoic acid signaling. Ultimately, the X. laevis cardiac response to KillerRed-induced oxidative stress shares characteristics with both mammalian and urodele/fish repair mechanisms, but is nonetheless a unique form of recovery, occupying an intermediate place on the spectrum of cardiac regenerative ability. An understanding of how Xenopus repairs cardiac damage can help bridge the gap between mammals and urodeles and contribute to new methods of treating heart disease.