Presence of ornithine decarboxylase antizyme in primary cultured hepatocytes of the frog Xenopus laevis

Ornithine decarboxylase (ODC; EC 4.1.1.17) could be induced in primary cultured hepatocytes of the frog, Xenopus laevis, by a hypotonic treatment. Addition of 10 mM putrescine caused a rapid decay of preinduced ODC after a lag period of 30 min. The putrescine-induced ODC decay was faster than the ODC decay in the presence of cycloheximide. Simultaneous addition of cycloheximide blocked the putrescine-induced acceleration of ODC decay, indicating an involvement of protein synthesis. Addition of putrescine to normal medium caused complete loss of ODC activity in 2 h and then ODC-inhibitory activity appeared and progressively increased. The inhibitory factor was non-dialysable and temperature-sensitive and showed a time-independent and stoichiometric pattern of ODC inhibition. On the basis of these observations the inhibitory factor was identified as ODC antizyme. These results indicated that in frog hepatocytes, like in mammalian cells and tissues, ODC is under negative feedback regulation mediated by antizyme.     

Regulation of estrogen receptors in primary cultured hepatocytes of the amphibian Xenopus laevis as estrogenic biomarker and its application in environmental monitoring

The present study aims to introduce the regulation of estrogen receptors (ER) in primary cultured hepatocytes of the amphibian Xenopus laevis as a further potential estrogenic biomarker. Time courses of free ER in cell cultures treated with 17beta-estradiol (E2), nonylphenol (NP), and bisphenol A (BPA) were determined by means of radioreceptorassay (RARA). All compounds led to an immediate drop of free ER followed by a significant increase. The estrogen specific induction of ER-mRNA in vitro during time course was verified by using semiquantitative RT-PCR demonstrating greatest differences after 36 h. Dose-response curves of ER-mRNA for E2, NP, and BPA revealed that E2 possessed highest estrogenicity starting at 10(-9) M, while NP and BPA induced significant increases at 10(-8) and 10(-7) M, respectively. Extracts of the river Alb were subjected to RARA for ER binding to cytosolic liver fraction as well as to primary cultured hepatocytes for assessment of ER-mRNA induction. The results by RARA demonstrated clearly that binding to ER was highest in sewage treatment plant effluents and increased during the course of the river. These findings could be correlated with induction of ER-mRNA levels in vitro indicating that both techniques are suitable for application in monitoring of estrogenic EDC.     

Stimulation of uridine phosphorylation in primary cultures of Xenopus laevis hepatocytes by estradiol-17 beta

The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity.     

Regulation of protein synthesis by estradiol 17 beta, dexamethasone and insulin in primary cultured Xenopus hepatocytes

Changes in the protein synthesis of Xenopus hepatocytes caused by insulin, estradiol-17 beta (estradiol) and dexamethasone were studied by using a primary culture in serum-free medium. All of these hormones stimulated the synthesis of secretory and intracellular proteins. Dexamethasone induced or stimulated the synthesis of many proteins (though limited in number), whereas estradiol induced or stimulated relatively few proteins, including the yolk precursor protein vitellogenin. The majority of these proteins differed in molecular weight and/or isoelectric point. When hepatocytes were treated with both steroids, most of the proteins were synthesized at the rates expected from the single treatment of the respective steroids. Thus, each steroid selectively stimulated the synthesis of its specific proteins. However, exceptional proteins were observed, whose syntheses were stimulated only by double treatment. In contrast, insulin seemed to cause an overall increase in individual secretory protein synthesis.     

Cyclic AMP phosphodiesterase activities in Xenopus laevis oocytes

Cyclic AMP phosphodiesterase activity has been identified in full-grown Xenopus oocytes in vivo and in vitro. About 50% of the in vitro phosphodiesterase activity was present in the solution fraction and 35% in a partially purified membrane fraction. Both activities exhibited high substrate affinity (Km about 10(-6) M). Sucrose gradient fractionation revealed two forms of phosphodiesterase: a 5 S form (peak I) and a 6.5 S form (peak II). Treatment with trypsin led to the activation of the soluble enzyme with the transformation of peak II into peak I. Ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, calcium dependent regulator, and Fluphenazine did not influence the enzyme activities suggesting that the oocyte phosphodiesterases were not Ca2+-dependent. Intact oocytes were induced to mature by exposure to progesterone; their phosphodiesterase activities and distribution tested in vitro were comparable to those of untreated oocytes.     

Development of the primary mouth in Xenopus laevis

The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.     

Characterization of proteolytic activities in embryos of Xenopus laevis

1. Proteolytic activities in early embryos of Xenopus laevis exhibited maximum levels at pH 3.2, 5.6 and 7.2 when 3H-BSA was used as substrate, and the maximum proteolytic activity at pH 3.2 was several thousand-fold higher during the tail bud stage than in the unfertilized egg. 2. The proteolytic activity at pH 3.2 was separated into two fractions by gel chromatography. One fraction corresponded to a mol. wt of about 40,000 and its activity was inhibited by thiol protease inhibitors. The other appeared to be a protease of much higher mol. wt. 3. The maximum activities at pH 5.6 and 7.2 appear to correspond to proteins of mol. wt greater than 1,000,000.     

Sialyltransferase activities in cultured rat hepatocytes

Previous studies on the age and sex dependency of the ganglioside patterns in rat liver in vivo and the concomitant determination of the activities of some enzymes involved in these pathways revealed the prominent role of the sialylation of GM3 to GD3 in determining the flow to the mono (a)- and polysialo (b)-series, respectively. Here, the influence of hormones on the activities of GM3 and GD3 synthases in isolated hepatocytes was studied. The combination of several factors (insulin, glucagon, epidermal growth factor, glucocorticoids) was found to be necessary for maintaining in vivo activity levels of GD3- but not of GM3-synthase.     

The primary structures and myotropic activities of two tachykinins isolated from the African clawed frog,Xenopus laevis

Two peptides with limited structural similarity to mammalian substance P (SP) and neurokinin A (NKA) have been isolated from extracts of the intestine of the African clawed frog (Xenopus laevis). The primary structure of an SP-like peptide was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH(2), which is identical to the previously characterized peptide, bufokinin isolated from the toad Bufo marinus. The primary structure of an NKA-related peptide was established as Thr-Leu-Thr-Thr-Gly-Lys-Asp-Phe-Val-Gly-Leu-Met.NH(2). Only the five amino acids at the C-terminal region of the peptide are identical to mammalian NKA whereas the N-terminal region shows no structural similarity to previously characterized tachykinins. Immunohistochemical investigations of the gut wall revealed a dense network of nerve fibres and nerve cell bodies containing SP/NKA-like substances. The myotropic effects of the Xenopus tachykinins were compared with the contractile effect of mammalian SP and NKA on isolated strips of circular smooth muscle from Xenopus stomach. No significant differences in potencies (-log EC(50)) or in intrinsic activities were observed between the Xenopus and mammalian peptides. The potencies for the Xenopus SP-like (8.49+/-0.15) and the NKA-like peptide (8.12+/-0.06) were similar suggesting that the amino acid sequence at the N-terminal region of the tachykinins is not important in activating the tachykinin receptors in Xenopus gastric smooth muscle. The maximum response to Xenopus SP (alpha=0.59+/-0.06) was significantly lower than to the NKA-like peptide (alpha=1.0) suggesting a more effective interaction of the NKA-like peptide with the tachykinin receptor(s) in Xenopus stomach.     

The effect of natural and synthetic antioxidant polyhydroxynaphthoquinones on cholesterol metabolism in cultured rabbit hepatocytes]

Primary cultures of rabbit hepatocytes were used to examine the effect of natural and synthetic antioxidants--polyhydroxynaphthoquinones (PHNQ) and alpha-tocopherol on cholesterol and bile acid synthesis. Histochrome, one of the PHNQ, slightly decreased cholesterol synthesis at concentrations 10-100 microM, whereas alpha-tocopherol stimulated cholesterol synthesis. After administration of histochrome or alpha-tocopherol into culture medium a significant stimulation of bile acid synthesis in dose-dependent manner was observed. The increase of bile acid secretion by histochrome in the presence of physiological concentration of HDL2 was found as well. Since histochrome in contrast to alpha-tocopherol enhanced accumulation of [14C] cholesterol of HDL2 in the hepatocytes, it was concluded that histochrome stimulated bile acid synthesis as a result of increased input of HDL2 cholesterol into hepatocytes. These data suggest that histochrome may exhibit a hypocholesterolemic effect by stimulation of bile acid synthesis and inhibition of cholesterol synthesis.     

Regenerative responses in cultured hindlimb stumps of larval Xenopus laevis.

The regenerative capacity of larval Xenopus laevis hindlimbs amputated through the tarsalia at different stages of development and explanted in vitro was tested. In the first experimental series hindlimb stumps from stage 53, 54, 55, and 57 larvae (according to Nieuwkoop and Faber, '56) were cultured in Leibovitz's L-15 medium supplemented with 10% FCS, and 0.04 U of insulin and 10(-8) mg of L-thyroxine per ml of medium. Results showed that the distal part of the limb stumps from stages 53, 54, and 55 formed a regeneration blastema composed of proliferating mesenchymal cells beneath a typical apical cap. No blastema was formed in the proximal part of the stump. In limb stumps from stage 57, a regeneration blastema did not form either in the proximal or in the distal part of the stump. In a second experimental series, hindlimb stumps from stage 55 larvae, denervated 5 days prior to amputation in order to eliminate any residual neurotrophic factor, were cultured in a simplified L-15 medium containing 2% FCS and lacking insulin and thyroxine. Results showed that also in these experimental conditions the stumps from stage 55 formed a conical regeneration blastema at the distal tip. The blastema cells duplicated their own DNA and divided. At the proximal extremity no regeneration blastema was formed. In the same culture medium, the stumps of larvae at stage 57 did not form a regeneration blastema.(ABSTRACT TRUNCATED AT 250 WORDS)     

XMam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos

Notch signaling is involved in cell fate determination and is evolutionally highly conserved in vertebrates and invertebrates. Mastermind is a nuclear protein which participates in Notch signaling and is involved in direct transactivation of target genes. Here we analyzed the expression and the function of Xenopus mastermind1 (XMam1) in the process of primary neurogenesis. XMam1 is 3,425 bp and encodes 1,139 amino acids. Overall, Mastermind proteins consist of a basic domain, two acidic domains and a glutamine-rich domain, which are highly conserved among species. The ubiquitous expression of XMam1 was observed in both maternal and zygotic stages. Whole-mount in situ hybridization showed that XMam1 mRNA was present in the ectoderm by the gastrula stage and localized at the anterior neural region in the neurula stage. Thereafter, XMam1 expression was restricted to the eye and otic vesicle in the tailbud-stage embryo. XMaml overexpression caused the repression of primary neural formation. The truncated form of XMam1 (lacking the C-terminus of XMam1; XMam1deltaC) led to excess formation of primary neurons. Furthermore, XMam1deltaC strongly repressed XESR-1 transactivation. These results show that XMaml is involved in primary neurogenesis by way of Notch signaling and is an essential component for transactivation of XESR-1 in Xenopus laevis embryos.     

Modulation of functional activities in cultured rat hepatocytes

Rat hepatocytes isolated by enzymatic dissociation of the liver must attach in order to survive for more than a few hours. In conventional culture conditions, they rapidly lose their highly differentiated functions, e.g. adult isozymic forms, enzyme response to specific hormones and cytochrome P-450-dependent monooxygenase activities. Incompletely differentiated cells such as perinatal and regenerating hepatocytes, can transiently exhibit a more differentiated state. Therefore, regulation of hepatic functions, particularly enzyme activities cannot be studied for more than a few days. Hepatocyte survival rate and maintenance of specific functions are dependent on nutrient composition of the medium as well as the substrate. Complex matrices, particularly that derived from the connective liver biomatrix, appear to have an important favorable effect. However, regardless of culture conditions specific functions cannot be quantitatively maintained for more than several days. Recent observations strongly suggest that such a problem may be overcome by mimicking in vivo specific cell-cell interactions. Thus when co-cultured with a liver epithelial cell line, probably derived from biliary ductular cells, adult hepatocytes remain able to synthesize high levels of albumin and to conjugate drugs. In these conditions, the cells secrete an abundant heterogeneous extracellular material. The co-cultures can be maintained in a serum-free medium and specific liver functions can be altered experimentally. Such a model could be appropriate for studying long-term induction and modulation of liver enzyme activities under defined experimental conditions.     

The expression of creatine kinase isozymes in Xenopus tropicalis,Xenopus laevis laevis,and their viable hybrid

Starch gel electrophoresis of creatine kinase (CK) isozymes of Xenopus tropicalis shows that at least two different genes code for CK in this diploid (2n=20) species. These genes seem to be orthologous to the CK-A and CK-C genes of extant crossopterygian fish. Additional isozymes may be interpreted either as products of duplicate genes or, more probably, as epigenetically modified forms of the homodimers A^tA^t and C^tC^t, respectively. The originally tetraploid species X. laevis laevis (2n=36), which may have arisen by hybridization of diploid ancestors some 30–40 million years ago, has retained expression of all duplicate CK-A and CK-C genes. Differential expression during ontogenesis (CK-A genes) and in different adult tissues (CK-C genes) indicates that divergence occurred not only with respect to the primary sequence of these duplicate genes, but also with respect to the regulation of their expression. In the interspecific hybrid X. 1. laevis × X. tropicalis, all parental CK genes appear to be expressed simultaneously in the heart. However, several subunit combinations cannot be detected on the zymograms.This work was supported by Swiss National Foundation for Scientific Research Grant 3.775.0.80.     

Localization of two IQGAPs in cultured cells and early embryos of Xenopus laevis

Mammalian IQGAP1 is considered to modulate organization of the actin cytoskeleton under regulation of signaling proteins Cdc42 or Rac and calmodulin [Bashour et al., 1997: J Cell Biol 137:1555-1566; Hart et al., 1996: EMBO J 15:2997-3005] and also to be involved in cadherin-based cell adhesion [Kuroda et al., 1998: Science 281:832-835]. However, its function in the cell has not been clear. In order to clarify the function of IQGAP, we investigated IQGAP in Xenopus laevis cells. We isolated two Xenopus cDNAs encoding homologues of mammalian IQGAP, XIQGAP1, and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. Immunofluorescent localization of XIQGAPs in Xenopus tissue cultured cells (XTC cells) and in developing embryos was examined. In XTC cells, XIQGAP1 was colocalized with F-actin at cell-to-cell contact sites, membrane ruffles in lamellipodia, and filopodia. During development of embryos, XIQGAP1 was concentrated in the borders of all embryonic cells. An intense staining for XIQGAP1 was found in regions undergoing active morphogenetic movements, such as the blastopore lip of gastrulae, and the neural plate, the notochord, and the somite of neurulae. These results suggest that XIQGAP1 is involved in both cell-to-cell adhesion and cell migration during Xenopus embryogenesis and in cultured cells. On the other hand, the localization of XIQGAP2 in XTC cells was distinct from that of XIQGAP1 although it was also seen in lamellipodia, filopodia, and borders between cells. In addition to these regions, strong nuclear staining was observed in both XTC cells and embryonic cells.     

Classification of DNA polymerase activities from ovaries of the frog,Xenopus laevis

Four distinct DNA polymerase activities were isolated from ovaries of the frog Xenopus laevis. Specific assays for each activity were established. The isolated activities were characterized by molecular weight, template-primer preferences, and sensitivity to specific inhibitors as Xenopus laevis ovarian DNA polymerases-α₁, -α₂, -β, and -γ. All previously described Xenopus laevis DNA polymerases were classified using these properties.     

Hypaxial muscle migration during primary myogenesis in Xenopus laevis.

In contrast to many vertebrates, the ventral body wall muscles and limb muscles of Xenopus develop at different times. The ventral body wall forms in the tadpole, while limb (appendicular) muscles form during metamorphosis to the adult frog. In organisms that have been examined thus far, a conserved mechanism has been shown to control migratory muscle precursor specification, migration, and differentiation. Here, we show that the process of ventral body wall formation in Xenopus laevis is similar to hypaxial muscle development in chickens and mice. Cells specified for the migratory lineage display an upregulation of pax3 in the ventro-lateral region of the somite. These pax3-positive cells migrate ventrally, away from the somite, and undergo terminal differentiation with the expression of myf-5, followed by myoD. Several other genes are selectively expressed in the migrating muscle precursor population, including neural cell adhesion molecule (NCAM), Xenopus kit related kinase (Xkrk1), and Xenopus SRY box 5 (sox5). We have also found that muscle precursor migration is highly coordinated with the migration of neural crest-derived melanophores. However, by extirpating neural crest at an early stage and allowing embryos to develop, we determined that muscle precursor migration is not dependent on physical or genetic interaction with melanophores.     

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.     

Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone

The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b5 contents progressively decreased throughout the 72-h culture period to less than 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching less than 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6 beta-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50-60% of initial values at 72 h. UDP-glucuronyltransferase activity towards digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione-S-transferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 microM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6 beta-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione-S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation. Furthermore, the use of DEX in cell culture medium to enhance cell survival does not maintain total drug-metabolizing enzyme capability, but appears to transiently and selectively increase expression of certain isozymes at the expense of others.