Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.     

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.     

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.     

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: functional coupling of the sphingosine-1-phosphate receptor to PLC-xbeta in Xenopus oocytes

We investigated the influence of the growth surface on the direction of Xenopus spinal neurite growth in the presence of a dc electric field of physiological magnitude. The direction of galvanotropism was determined by the substratum; neurites grew toward the negative electrode (cathode) on untreated Falcon tissue culture plastic or on laminin substrata, which are negatively charged, but neurites growing on polylysine, which is positively charged, turned toward the positive electrode (anode). Growth was oriented randomly on all substrata without an electric field. We tested the hypothesis that the charge of the growth surface was responsible for reversed galvanotropism on polylysine by growing neurons on tissue culture dishes with different net surface charges. Although neurites grew cathodally on both Plastek substrata, the frequency of anodal turning was greater on dishes with a net positive charge (Plastek C) than on those with a net negative charge (Plastek M). The charge of the growth surface therefore influenced the frequency of anodal galvanotropism but a reversal in surface charge was insufficient to reverse galvanotropism completely, possibly because of differences in the relative magnitude of the substratum charge densities. The influence of substratum adhesion on galvanotropism was considered by growing neurites on a range of polylysine concentrations. Growth cone to substratum adhesivity was measured using a blasting assay. Adhesivity and the frequency of anodal turning were graded over the range of polylysine concentrations (0 = 0.1 < 1 < 10 = 100 microg/ml). The direction of neurite growth in an electric field is therefore influenced by both substratum charge and growth cone-to-substratum adhesivity. These data are consistent with the idea that spatial or temporal variation in the expression of adhesion molecules in embryos may interact with naturally occurring electric fields to enhance growth cone pathfinding.     

Characterization and subcellular localization of ribonuclease H activities from Xenopus laevis oocytes

Ribonuclease H activities present in fully grown Xenopus oocytes were investigated by using either liquid assays or renaturation gel assays. Whereas the test in solution detected an apparently unique class I ribonuclease H activity, the activity gels did not detect this enzyme but another one with the molecular weight expected for a class II ribonuclease H. The ribonuclease HI was found to be primarily concentrated in the germinal vesicle, but around 5% of this activity was detectged in the cytoplasm and may correspond to the activity involved in antisense oligonucleotide-mediated destruction of messenger RNAs. The concentration of this class I ribonuclease H in oocytes is similar to that in somatic cells. The class II ribonuclease H remained undetectable by the test in solution because its activity was cryptic. On activity gel, a polypeptide with the apparent molecular mass of 32 kDa, expected for a ribonuclease HII, was found to be concentrated in mitochondria although no RNase H activity could be detected by using the liquid assay. Based on sedimentation studies, we hypothesize that the apparent absence of RNase H activity in solution could be the result of the association of this 32-kDa polypeptide with other polypeptides, or possibly nucleic acids, to form a multimer of, until now, unknown function.     

Nucleolar localization elements of Xenopus laevis U3 small nucleolar RNA

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected into Xenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5' region containing Boxes A and A', known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5' cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box C' led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing.     

Cellular localization of dopamine D2 receptor messenger RNA in the rat trigeminal ganglion

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.     

Improved preparation of Xenopus oocytes for patch-clamp recording

The testis-determining gene SRY (sex determining region, Y) is located on the short arm of the Y chromosome and consists of a single exon, the central third of which is predicted to encode a conserved motif with DNA binding/bending properties. We describe the screening of 26 patients who presented with 46,XY partial or complete gonadal dysgenesis for mutations in both the SRY open reading frame (ORF) and in 3.8 kb of Y-specific flanking sequences. DNA samples were screened by using the fluorescence-assisted mismatch analysis (FAMA) method. In two patients, de novo mutations causing complete gonadal dysgenesis were detected in the SRY ORF. One was a nonsense mutation 5' to the HMG box, whereas the other was a missense substitution located at the C terminus of the conserved motif and identical to one previously detected in an unrelated patient. In addition, two Y-specific polymorphisms were found 5' to the SRY gene, and a sequence variant was identified 3' to the SRY polyadenylation site. No duplications of the DSS region in 20 of these patients were detected.     

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.     

Leucine transport in Xenopus laevis oocytes: functional and morphological analysis of different defolliculation procedures

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na(+)-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol oocyte-1 40 min-1 in folliculated oocytes and 5.6 +/- 1.9 in collagenase defolliculated oocytes (means +/- SE). L-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled L-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine binding to membranes. L-leucine kinetics showed that the L-leucine Vmax and Km values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The Vmax and Km values of Na(+)-dependent L-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16 +/- 1.5 pmol oocyte-1 40 min-1 and 57 +/- 21 mumol (mean +/- SD). The Na(+)-activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of L-leucine Vmax, and microscopic observation showing recovery of the microvillar apparatus.     

Galpha14 and Galphaq mediate the response to trypsin in Xenopus oocytes

Xenopus oocytes respond to trypsin with a characteristic chloride current, virtually indistinguishable from responses mediated by a large number of native and expressed G protein-coupled receptors. We studied the involvement of G proteins of the Galphaq family as possible mediators of this and other G protein-coupled receptor-mediated responses in Xenopus oocytes. We have cloned the third member of the Galphaq family, Xenopus Galpha14, in addition to the previously cloned Xenopus Galphaq and Galpha11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89-92). Amphibian Galpha14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the Galpha14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiGalpha14) that, together with antiGalphaq and antiGalpha11, was used in antisense depletion experiments. 24 h after injection into oocytes, either antiGalphaq or antiGalpha14 reduced the response to 1 microg/ml trypsin by 70%, whereas antiGalpha11 had no effect. A mixture of antiGalphaq and antiGalpha14 virtually abolished the response. These data strongly suggest that Galphaq and Galpha14 are the exclusive mediators of the trypsin-evoked response in Xenopus oocytes. Similar experiments with the expressed gastrin-releasing peptide receptor and muscarinic m1 receptor revealed the coupling of Galphaq and Galpha11, but not Galpha14, to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the Galphaq family discriminate among different native receptors in vivo.     

Defined nutrient medium for the in vitro maintenance of Xenopus laevis oocytes

A procedure is described for the isolation and culture of large numbers of follicle cell-free Xenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenace of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morpological and functional characteristics of oocytes for periods up to 3 weeks.     

GTP analogs suppress uptake but not transport of D-glucose analogs in Glut1 glucose transporter-expressing Xenopus oocytes

The effect of electrical stimulation of the ventroposterolateral (VPL) thalamic nucleus on mechanical allodynia in the unrestrained awake rat was investigated. In 7 rats, a monopolar stimulation and recording electrode was implanted in the VPL thalamic representation area of the hindpaw. Exact target localisation was performed by means of thalamic evoked potentials induced by stimulating the contralateral tibial nerve. A peripheral mononeuropathy was induced by partly ligating the right sciatic nerve. Sensitivity of the hindpaws to mechanical stimulation was assessed with a set of von Frey hairs. One to 4 weeks after nerve ligation, all rats showed allodynia to mechanical stimulation and signs of spontaneous pain. Electrical stimulation of the contralateral VPL thalamic nucleus abolished the mechanical allodynia observed at the nerve ligated side. The effect of VPL stimulation outlasted the stimulation period by 15 min. No effect on the withdrawal thresholds at the control (sham operated) side was observed. These animals data support the clinical reports that stimulation of the sensory thalamus may alleviate pain of neuropathic origin.     

Lithium homeostasis in Xenopus oocytes: implications for the study of signal transduction

The Xenopus oocyte has been shown to be a useful model for the study of signal transduction pathways. The present study investigated whether or not the oocyte could be used to study the effects of lithium on signal transduction mechanisms by comparing the dynamics of lithium homeostasis in the oocyte and a human immortalized hippocampal cell line using Flame Atomic Emission Spectroscopy (FAES). A biphasic pattern of lithium uptake was observed in the oocyte in the presence of 5 mM extracellular lithium. The late phase of lithium uptake, which started after 30 minutes of incubation time, was sensitive to phloretin, an inhibitor of Na+/Li+ counter-transport. Differences in lithium efflux kinetics further characterized the two observed phases of accumulation and also suggested that lithium might be distributed in different pools within the oocyte, including one sequestered in organelles or associated with cytosolic proteins. An analogous sequestered pool was not, however, observed in the hippocampal cell line indicating that lithium is distributed differently in these cell types. This suggests that the Xenopus oocyte might not be a suitable model for evaluating the effects of lithium on signal transduction pathways because of the unknown contribution of the sequestered pool on predicting relevant physiological effects.