Identification of a multixenobiotic resistance mechanism in Xenopus laevis embryos

In the '90's a membrane-associated transport protein, discovered in aquatic organisms, was considered to be expressed in response to environmental xenobiotics. Like the multidrug resistance protein found in mammalian tumor cell lines, this protein confers resistance in organisms in polluted areas by binding xenobiotics and transporting them out of the cells in an energy-dependent manner. This study investigates the expression and the activity of a P-glycoprotein (Pgp) involved in a multixenobiotic resistance mechanism (MXRM) during the early developmental stages and in tissues of adult Xenopus laevis.     

The effect of concanavalin-A on the reaggregation of cells dissociated from Xenopus laevis early embryos

The effects of concanavalin-A on the reaggregation and sorting of cells from Xenopus laevis early embryos have been studied. The results suggest that at high concentrations, concanavalin-A can prevent reaggregation.     

Ribosomes from Xenopus laevis eggs and embryos in a cell-free protein-synthesizing system: translational regulation

Three types of ribosomal preparations from Xenopus laevis eggs and embryos were tested in a cell-free system to study possible translational regulation of protein synthesis as mediated by the ribosome during early amphibian development: type 1, a crude high-speed sediment, mainly containing monoribosomes completely dissociable by 0.5 M KC1; type II, ribosomes washed with 0.5 M KC1; and type III, ribosomes treated with puromycin - 0.5 M KC1. All three types showed an active response to the addition of poly[U]. Type III was found to be the most active: levels of incorporation of 30 phenylalanine residues/ribosome were reached. In all three cases ribosomes prepared from unfertilized eggs were 30-40% less active in vitro than those from cleavage and gastrula stages.     

Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells

A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.     

Follicular tissues reduce drug effects on ion channels in oocytes of Xenopus laevis

The influence of follicular tissues on drug effects on ion channels in Xenopus oocytes was tested by investigating the pharmacological properties of a cloned potassium channel in oocytes with and without follicular tissues. The data show that the efficacy of blocking agents (ranging from metal ions to peptides) is drastically reduced by the follicular tissues (reductions by as much as 90% and increases of the IC50 values up to 30-fold). Furthermore, the time course of the blocking effect was slowed down by the tissues (increases of the t50 values up to 40-fold). The described impairment could be mitigated, but not abolished by partial removal of the follicular tissues (so-called defolliculation, leaving only the vitelline envelope and part of the follicle cells on the oocyte surface). The results indicate that the follicular tissues can induce significant errors in pharmacological measurements on membrane proteins in Xenopus oocytes.     

pXen, a utility vector for the expression of GST-fusion proteins in Xenopus laevis oocytes and embryos

We describe a plasmid, pXen, designed for the optimized expression of proteins fused to glutathione-S-transferase (GST) in Xenopus laevis oocytes and embryos. The Xenopus model system permits the biochemical analysis of signaling pathways and analysis of embryo phenotype in response to manipulation of proto-oncogene expression. pXen is a modified pSP64T vector which contains an SP6 RNA polymerase promoter followed by the translational initiation sequence of Xenopus beta-globin and the glutathione binding domain of GST. The Xenopus 3' beta-globin untranslated region and polyadenylation site immediately follow the multiple cloning site to permit the efficient translation of in vitro transcribed RNA in oocytes and embryos. The utility of pXen is demonstrated by cloning the catalytic domain of the serine/threonine kinase proto-oncogene Raf-1 into this vector and injecting the corresponding in vitro transcribed RNA into oocytes. Catalytically active GST-vRaf fusion protein was expressed in the injected oocytes and induced oocyte maturation. Moreover, the GST-vRaf fusion protein could be readily purified from Xenopus extracts using glutathione Sepharose. We demonstrate that the Raf-1 catalytic domain retains activity when fused with the N-terminal GST moiety and is subject to negative regulation by the cyclic AMP-dependent protein kinase (PKA). The pXen vector will be useful for an in vivo analysis of the physiological role and regulation of a wide variety of signaling molecules when expressed in Xenopus oocytes and embryos.     

Induction of mortality and malformation in Xenopus laevis embryos by water sources associated with field frog deformities

Water samples from several ponds in Minnesota were evaluated for their capacity to induce malformations in embryos of Xenopus laevis. The FETAX assay was used to assess the occurrence of malformations following a 96-hr period of exposure to water samples. These studies were conducted following reports of high incidences of malformation in natural populations of frogs in Minnesota wetlands. The purpose of these studies was to determine if a biologically active agent(s) was present in the waters and could be detected using the FETAX assay. Water samples from ponds with high incidences of frog malformations (affected sites), along with water samples from ponds with unaffected frog populations (reference sites), were studied. Initial experiments clearly showed that water from affected sites induced mortality and malformation in Xenopus embryos, while water from reference sites had little or no effect. Induction of malformation was dose dependent and highly reproducible, both with stored samples and with samples taken at different times throughout the summer. The biological activity of the samples was reduced or eliminated when samples were passed through activated carbon. Limited evidence from these samples indicates that the causal factor(s) is not an infectious organism nor are ion concentrations or metals responsible for the effects observed. Results do indicate that the water matrix has a significant effect on the severity of toxicity. Based on the FETAX results and the occurrence of frog malformations observed in the field, these studies suggest that water in the affected sites contains one or more unknown agents that induce developmental abnormalities in Xenopus. These same factors may contribute to the increased incidence of malformation in native species.     

Depressant effects of volatile anesthetics on second messenger system in mRNA-expressed Xenopus laevis oocytes

In this study I attempted to elucidate the depressant effects of volatile anesthetics on inositol trisphosphate (IP3)-mediated signal transduction pathway and to identify the site of action. For this purpose, we used Xenopus laevis oocytes which translated and expressed 5-HT receptors after injection of mRNA isolated from the rat brain. In this system, binding of the agonist to G-protein coupled receptors activates phospholipase C that produces IP3. Mobilization of Ca2+ by IP3 from the storage finally opens Ca2+ dependent Cl- channels. Halothane, isoflurane and methoxyflurane depressed Cl- current elicited by 5-HT. For the further quantitative study, methoxyflurane was used because of its better solubility and less vapor pressure that avoided evaporation of the agent. The 5-HT elicited Cl- current was depressed in a non-competitive fashion. Response were 75, 60, 20% of control in the presence of 0.5, 1 and 3 mM methoxyflurane, respectively. Responses elicited by a pressure-injection of Ca2+ or IP3 remained unchanged in the presence of high concentrations of either halothane, isoflurane or methoxyflurane. These results suggest that the depressant mechanism by volatile anesthetics on the signal transduction pathway involves neither Ca2+ dependent Cl- channel dynamics nor intracellular Ca2+ mobilization by IP3. Changes of microdomain characteristics of the membrane in the presence of anesthetic molecules including membrane-bound proteins and enzyme system may be a main mechanism of action of volatile anesthetics.     

X-twi is expressed prior to gastrulation in presumptive neurectodermal and mesodermal cells in dorsalized and ventralized Xenopus laevis embryos

Interactions between cytokines and Schwann cells (SC) are important in development, repair, and disorders of the peripheral nervous system (PNS). Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) are two prominent cytokines which may be involved in these processes and their gene products are upregulated in some experimental neuropathies. This study focuses on the in vitro effects of these cytokines, both singly and in combination, on cultured SC. Expression of both Type I and Type II TNF-alpha receptors was demonstrated on the SC surface by immunocytochemistry. Treatment of SC with a combination of TNF-alpha plus TGF-beta causes significant detachment and cell death while treatment with each cytokine alone is not significantly cytotoxic. When compared with control cultures, SC treated with the combination of cytokines exhibit an increase in the number of cells with condensed nuclei and evidence of DNA fragmentation, characteristics consistent with cells undergoing programmed cell death. Thus, TNF-alpha plus TGF-beta induce SC loss of adhesion which is predominantly due to cell death. Apoptotic mechanisms are likely to contribute to some extent to this cell death. These findings provide in vitro evidence to support the hypothesis that cytokines can directly damage SC in PNS disorders.     

Observations on the migration and proliferation of gonocytes in Xenopus laevis

The process of primordial germ cell formation in the normal course of development of Xenopus laevis was examined with a light microscope on paraffin and Epon sections of embryos or tadpoles, extending over the period from the gastrula to the feeding tadpole stage. Positional changes of gonocytes with development were nearly the same as those reported on the same species by Blackler (1958) and Whitington & Dixon (1975). The following points were newly demonstrated. Gonocytes which have been located in a deep endodermal position till mid tail-bud stage come to be located in a rather peripheral region of the endoderm cell mass at stage 31 (late tail-bud), suggesting that the initial step of migration of the gonocytes towards the future genital ridge has already begun at this stage. Gonocytes at stages 33/34 and 35/36 were observed in a more dorsal part of the endoderm than at stage 31. Gonocytes which seem to have begun their migration are roundish in external shape and have a large intercellular space around them. At stage 40 gonocytes were located in the dorsal endodermal crest, and at stage 41 gonocytes were found with cell bodies extending over both the dorsal endoderm crest and the dorsal mesentery, indicating that the separation of the gonocytes from the endoderm was in progress at this stage. The present results seem to indicate that gonocytes migrate not passively but actively from the deep endodermal position to the genital ridge, passing through the dorsal mesentery. Counting the number of gonocytes at successive stages of development revealed that gonocytes proliferated exponentially throughout the developmental stages from gastrula to tadpole.     

Development of the gut in Xenopus laevis

The lining of the gut, together with the pancreas, liver, gall bladder, and respiratory system, is formed from the endoderm. The gut also contains smooth muscle and connective tissue of mesodermal origin. The amphibian Xenopus laevis is potentially an excellent model organism for studying how the cells of the endoderm and mesoderm become programmed to produce these internal organs. However, the anatomical complexity of the coiled gut presents a problem in studying its development. In order to overcome this problem we here present a comprehensive guide to the anatomy and histology of the developing Xenopus gut. We use a simple dissection to display its anatomy and the expression of four endodermal markers (alkaline phosphatase, IFABP, XlHbox8, and endodermin). We present schematic diagrams that show how the gut is arranged in three dimensions and how this organisation changes during development. We also present drawings of histological sections of the gut which allow any region to be identified and so represent an atlas for working with sections. Finally, we describe the histology of the cells of the various organs of the gut. This histological identification may be necessary for the identification of parts following experiments in which the normal pattern is disturbed.     

Development of the interphotoreceptor matrix in Xenopus laevis

Xenopus laevis interphotoreceptor matrix (IPM) contains a relatively aqueous insoluble wheat germ agglutinin (WGA)-binding component containing unidentified sialoglycoconjugates (Wood et al [1984] J. Comp. Neurol. 228:299-307). The appearance of WGA-binding macromolecules in the IPM was assessed during late embryonic stages (32-45) and in retinal rudiment cultures, using lectin cytochemistry and Western blotting techniques. Metabolic labeling of the neural retina versus retinal pigment epithelium (RPE)-choroid of juvenile Xenopus with 35S-MET was also evaluated in vivo and in vitro. Lectin cytochemistry of eyes from developmental stages 32-42 demonstrated distinct WGA-ferritin-binding sites on the developing outer segment membranes and in the IPM compartment. At stages 44-46 extensive WGA-binding domains were present as an extracellular network with other randomly scattered domains near the retinal pigment epithelium. Retinal rudiments from stage 32-33 were isolated and allowed to differentiate in hanging drop culture (Hollyfield and Witkowsky [1974] J. Exp. Zool. 189:357-377) with or without an investing pigment epithelium. Cultures developing with RPE exhibited an elaborate IPM with an anastomosing meshwork of WGA-ferritin binding sites. In the absence of RPE only limited amounts of binding restricted to the immediate vicinity of the developing photoreceptor outer segment membranes was observed. When Western blots were probed with WGA-HRP, stage 32-45 retinas demonstrated a major WGA-binding band of 126 kD. Similar amounts of WGA-binding macromolecules were synthesized in preparations cultured in the presence or absence of the investing RPE. During development the major WGA-binding component is a 126-kD protein. Equivalent synthesis of this protein in the presence and absence of RPE suggests that the PE is not required for synthesis of this 126-kD component. These results suggest that the retina is the primary site of synthesis of the WGA-binding components of the Xenopus IPM, whereas the PE plays a principal role in their assembly and organization.     

Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of XLerk, an erythropoietin-producing hepatocellular ligand

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a Xenopus homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of Xenopus embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal 19 amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic fibroblast growth factor, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and fibroblast growth factor signaling modulates this function.     

Purification and some properties of ribonuclease from Xenopus laevis eggs

A 122 kDa RNase from eggs of Xenopus laevis was purified by sequential chromatography on Sephadex G-75, DEAE-cellulose, heparin-Sepharose and TSK gel G3000SW columns, and gave a single 60 kDa band on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The RNase composed of two 60 kDa subunits is able to recognize pyrimidine bases specifically. The pH optimum of the RNase was 7.5 in Tris-HCl buffer. The enzyme activity was abolished by treatment at 80 degrees C for 5 min and pH 2 or 12 for 1 h. Since egg lectins with RNase activity obtained from Rana catesbeiana and R. japonica and bovine pancreatic RNase A show about 30% protein homology and these three proteins are 12-14 kDa heat-stable RNases, [K. Titani, K. Takio, M. Kuwada, K. Nitta, F. Sakakibara, H. Kawauchi, G. Takayanagi and S. Hakomori, Biochemistry, 26, 2189 (1987); Y: Kamiya, F. Oyama, R. Oyama, F. Sakakibara, K. Nitta, H. Kawauchi, Y. Takayanagi and K. Titani, J. Biochem. (Tokyo), 108, 139 (1990)], the data suggest that the X. laevis egg RNase is a unique protein compared with RNases from not only amphibians, but also mammals.     

The deleterious effects of exogenous angiotensin I and angiotensin II on myocardial ischemia-reperfusion injury

Angiotensin II is well known to have a cardiotoxic effects. However, it is still unclear whether exogenous angiotensin I or angiotensin II has a deleterious effect on myocardial ischemia-reperfusion injury. To examine this deleterious effects, we administered angiotensin I and angiotensin II to perfused hearts before ischemia, and measured creatine kinase (CK) release and cardiac function during subsequent reperfusion. Wistar Kyoto rats were used and the hearts were perfused by the Langendorff technique at a constant flow (10 ml/min). Seven hearts were perfused for 20 min and then subjected to 15 min of global ischemia (Control). In the experimental groups, during the 5 min before ischemia, we administered 100 ng/ml angiotensin I (Ang I; n = 9), 1 microgram/ml enalaprilat (ACEI; n = 5), both agents (ACEI + Ang I) (n = 6), or 10 ng/ml angiotensin II (Ang II; n = 6). The perfusates were then sampled to measure angiotensin II. After 15 min of ischemia, the hearts were reperfused with control perfusate. Throughout the 20 min of reperfusion, the effluent was collected to measure cumulative CK release. Angiotensin I increased coronary perfusion pressure (CPP) by 32 +/- 4 mmHg, however, the angiotension converting enzyme inhibitor inhibited the increase of CPP by angiotension I (11 +/- 1 mmHg) (p < 0.01). The contents of angiotensin II in the effluent in Ang I and Ang I + ACEI were 11.5 +/- 1.9 ng/ml and 4.0 +/- 0.5 ng/ml (p < 0.01). After 20 min of reperfusion, the left ventricular developed pressure was unchanged in all of the groups. CPP was also unchanged by ischemia in all of the groups.(ABSTRACT TRUNCATED AT 250 WORDS)