Lipovitellin 2β is the 31 kD Ni2+-binding protein (pNiXb) in Xenopus oocytes and embryos

An Ni²⁺-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with ⁶³Ni²⁺, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2β, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2β agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metalblot competition assay, the abilities of metal ions to compete with ⁶³Ni²⁺ for binding to pNiXb were ranked: Zn²⁺ ≈ Cu²⁺ ≈ Co²⁺ > Cd²⁺ ≈ Mn²⁺ > Sn²⁺. This study shows that Xenopus lipovitellin 2β is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo. © 1994 Wiley-Liss, Inc.     

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca²⁺ channel, in store-operated Ca²⁺ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3 and 5 rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-1 polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca²⁺ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP₃F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC₅₀ = 0.5 M), indicating that InsP₃F and LPA principally activate store-operated Ca²⁺ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP₃F-, LPA- or thapsigargin-stimulated Ca²⁺ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP₃F-stimulated Ca²⁺ inflow and only a very small enhancement of LPA-stimulated Ca²⁺ inflow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca²⁺ inflow through the previously-characterised Ca²⁺-specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Tamarix hispida metallothionein-like ThMT3, a reactive oxygen species scavenger, increases tolerance against Cd2+, Zn2+, Cu2+, and NaCl in transgenic yeast

A metallothionein-like gene, ThMT3, encoding a type 3 metallothionein, was isolated from a Tamarix hispida leaf cDNA library. Expression analysis revealed that mRNA of ThMT3 was upregulated by high salinity as well as by heavy metal ions, and that ThMT3 was predominantly expressed in the leaf. Transgenic yeast (Saccharomyces cerevisiae) expressing ThMT3 showed increased tolerance to Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress. Transgenic yeast also accumulated more Cd²⁺, Zn²⁺, and NaCl, but not Cu²⁺. Analysis of the expression of four genes (GLR1, GTT2, GSH1, and YCF1) that aid in transporting heavy metal (Cd²⁺) from the cytoplasm to the vacuole demonstrated that none of these genes were induced under Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress in ThMT3-transgenic yeast. H₂O₂ levels in transgenic yeast under such stress conditions were less than half those in control yeast under the same conditions. Three antioxidant genes (SOD1, CAT1, and GPX1) were specifically expressed under Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress in the transgenic yeast. Cd²⁺, Zn²⁺, and Cu²⁺ increased the expression levels of SOD1, CAT1, and GPX1, respectively, whereas NaCl induced the expression of SOD1 and GPX1.     

Involvement of Ca2+-calmodulin in Cd2+ toxicity during the early phases of radish (Raphanus sativus L.) seed germination

The toxicity of Cd²⁺in vivo during the early phases of radish (Raphanus sativus L.) seed germination and the in vitro Cd²⁺ effect on radish calmodulin (CaM) were studied. Cd²⁺ was taken up in the embryo axes of radish seeds; the increase in fresh weight of embryo axes after 24 h of incubation was inhibited significantly in the presence of 10 mmol m^?3 Cd²⁺ in the external medium, when the Cd²⁺ content in the embryo axes was c. 1.1 μmol g^?1 FW. The reabsorption of K⁺, which characterizes germination, was inhibited by Cd²⁺, suggesting that Cd²⁺ affected metabolic reactivation. The slight effect of Cd²⁺ on the transmembrane electric potential of the cortical cells of the embryo axes excluded a generalized toxicity of Cd²⁺ at the plasma membrane level. After 24 h of incubation, Cd²⁺ induced no increase in total acid-soluble thiols and Cd²⁺-binding peptides able to reduce Cd²⁺ toxicity. Ca²⁺ added to the incubation medium partially reversed the Cd²⁺-induced inhibition of the increase in fresh weight of embryo axes and concomitantly reduced Cd²⁺ uptake. Equilibrium dialysis experiments indicated that Cd²⁺ bound to CaM and competed with Ca²⁺ in this binding. Cd²⁺ inhibited the activation of Ca²⁺-CaM-dependent calf-brain phosphodiesterase, inhibiting the Ca²⁺-CaM active complex. Cd²⁺ reduced the binding of CaM to the Ca²⁺-CaM binding enzymes present in the soluble fraction of the embryo axes of radish seeds. The possibility that Cd²⁺ toxicity in radish seed germination is mediated by the action of Cd²⁺ on Ca²⁺-CaM is discussed in relation to the in vivo and in vitro effects of Cd²⁺.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Cd2+-induced damage to yeast plasma membrane and its alleviation by Zn2+: studies on Schizosaccharomyces pombe cells and reconstituted plasma membrane vesicles

In Schizosaccharomyces pombe, Cd²⁺ shares the same uphill uptake system with Zn²⁺. Both heavy metals inhibited growth, respiration, H⁺/glucose uptake, and glucose-induced proton extrusion, Cd²⁺ being a 10–15-fold stronger inhibitor. In contrast, both had a similar effect on the plasma membrane H⁺-ATPase, enhancing its affinity for ATP and reducing the rate of ATP splitting. Cd²⁺ caused protracted strong fluidization of the plasma membrane of energized cells, whereas deenergized cells, phosphatidylcholine liposomes, and plasma membrane fragments, either purified or incorporated into the liposomes, exhibited only a short initial fluidization. Zn²⁺, which caused only a marginal membrane fluidization, suppressed the fluidizing action of Cd²⁺. The fluidizing effect of both heavy metals on liposomes was reduced by the presence of plasma membrane fragments in the liposome membrane. At 50 μM, Cd²⁺ brought about loss K⁺ (18 K⁺/1 Cd²⁺) from energized, but not from deenergized cells since Cd²⁺ must first accumulate in the cells before causing a detectable effect. A simple membrane disruption by external Cd²⁺ is, therefore, unlikely to be the main mechanism of cadmium-induced potassium loss in intact cells. Zn²⁺ had virtually no effect below 1 mM concentration, and it again weakened the K⁺-releasing effect of Cd²⁺. Cd²⁺ caused a strong loss of K⁺ also from K⁺-containing liposomes, probably because of a direct interaction with liposome phospholipids. Incorporation of plasma membrane fragments into the liposomes reduced the K⁺ loss sixfold. Received: 13 November 1995 / Accepted: 31 January 1996     

Protein synthesis in interspecies hybrid embryos of the amphibian Xenopus

The newly synthesized abundant proteins of early Xenopus laevis and Xenopus borealis embryos have been examined by two-dimensional electrophoresis after labelling with [³⁵S]methionine. Six prominent polypeptides specific to Xenopus laevis embryos and a further six specific to Xenopus borealis have been identified. Overall, embryos of the two species are estimated to differ by approx. 15% in their protein synthetic patterns from blastula to tailbud stage. Interspecific hybrid embryos (Xenopus laevis (♀)/Xenopus borealis (♂)) synthesise only the maternally specified set of proteins until gastrulation after which they produce the full complement of both Xenopus laevis and Xenopus borealis specific proteins. The possible use of such molecular markers of parental genome activity in facilitating further embryological study is discussed.     

In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes

In vitro selection of Zn²⁺-dependent RNA-cleaving DNAzymes with activity at 90°C has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn²⁺ over Pb²⁺, Ca²⁺, Cd²⁺, Co²⁺, Hg²⁺, and Mg²⁺. The Zn²⁺-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn²⁺-binding dissociation constant of 234 μM and that its activity increases with increasing temperatures from 50–90°C. A fit of the Arrhenius plot data gave E_a = 15.3 kcal mol⁻¹. Surprisingly, the selected Zn²⁺-dependent DNAzymes showed only a modest (∼3-fold) activity enhancement over the background rate of cleavage of random sequences containing a single embedded ribonucleotide within an otherwise DNA oligonucleotide. The result is attributable to the ability of DNA to sustain cleavage activity at high temperature with minimal secondary structure when Zn²⁺ is present. Since this effect is highly specific for Zn²⁺, this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.     

Uptake and release of63Ni2+ byXenopus embryos during early cleavage stages

Summary Uptake and release of⁶³Ni was studied in dejelliedXenopus laevis embryos exposed to⁶³Ni²⁺ (0.3–30 mol/1) for 0.5-h intervals during the period 1–4.5 h post-fertilization (i.e. from first cleavage to early blastula stage). At first cleavage, the mean uptake of⁶³Ni by embryos was 12-17 times that by non-fertilized eggs, suggesting that conversion of the vitelline envelope to the fertilization envelope enhanced integumental permeability to⁶³Ni²⁺. 63 Ni uptake by embryos at the 1-2-cell stage averaged 1.8–2.5 times that at the early blastula stage. An average of 5% of total⁶³Ni in washed embryos was recovered in isolated fertilization envelopes, indicating that⁶³Ni²⁺ passed through the envelope into internal compartments. Progressive increases of⁶³Ni uptake were seen with increasing exposure levels; after exposure during 1–1.5 h post-fertilization to the highest concentration of⁶³Ni²⁺ (30 mol/1),⁶³Ni uptake averaged 11.4 (SD±5.1) pmol/embryo. Rapid efflux of⁶³Ni was noted after⁶³Ni²⁺-exposed embryos were transferred to nickel-free medium; mean⁶³Ni contents at 0.25 h and 2 h post-exposure diminished to 50% and 15% of the initial values, regardless of the exposure level. The finding thatXenopus embryos are permeable to⁶³Ni²⁺ during early cleavage stages provides a convenient experimental system to investigate the embryotoxicity and teratogenicity of nickel.     

Inhibition kinetics of brain butyrylcholinesterase by Cd2+ and Zn2+, Ca2+ or Mg2+ reactivates the inhibited enzyme

• 1.1. The inhibition kinetics of sheep brain butyrylcholinesterase (BChE) (acylcholine acylhydrolase, EC 3.1.1.8) by Cd²⁺ and Zn²⁺ has been studied.
• 2.2. K_s has been determined as 0.14mM. Cd²⁺ and Zn²⁺ were the hyperbolic mixed-type inhibitors of BChE. Ca²⁺ and Mg²⁺ had no effect on the enzyme activity in the experimental conditions.
• 3.3. But when the enzyme was inhibited by 0.1 mM Cd²⁺ or Zn²⁺, Ca²⁺ and Mg²⁺ reactivated the inhibited form of BChE.

     

Vitellogenesis in Bufo arenarum: Identification,characterization and immunolocalization of high molecular mass lipovitellin during oogenesis

Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late).     

Synthesis and processing of the mouse MOPC-321 kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from mineral oil-induced plasmacytoma (MOPC)-321 was injected into Xenopus laevis oocytes that were incubated in ³H-labeled amino acids. The MOPC-321 k chain was purified from an oocyte homogenate by immunoprecipitation, followed by preparative gel electrophoresis. To determine whether the precursor segment had been properly and precisely cleaved in the oocyte, the amino acid sequence of the NH₂terminal end of the purified k chain was investigated. The NH₂-terminal sequence obtained was identical to that of the mature, secreted form of the protein. Thus the specificity of the enzyme performing the cleavage of precursor to mature chain is similar in frog oocytes and in mammalian cells. Therefore, the enzymatic specificity has been highly conserved during evolution and evidently performs an essential role in cellular metabolism.     

Mitochondrial DNA synthesis in oocytes of Xenopus laevis

Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35 ± 0.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28 ± 0.02 pg/oocyte/h. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [³H]-dTTP was calculated to be 0.43 ± 0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.     

重金属铜、锌、镉复合胁迫对麻疯树幼苗生理生化的影响

该研究以Cu~(2+)、Zn~(2+)、Cd~(2+)单一胁迫为对照,探讨不同浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫对麻疯树幼苗生理生化指标的影响。结果表明:随着Cu~(2+)、Zn~(2+)、Cd~(2+)浓度的增加,麻疯树幼苗叶片中的蛋白质(Pro)、丙二醛(MDA)含量均逐渐增加,其叶片叶绿素含量随着Zn~(2+)胁迫浓度的增加呈现出先降后升的趋势,在中等浓度(100 mg·L-1)的Zn~(2+)胁迫时含量最低、随着Cu~(2+)胁迫浓度的增加叶绿素含量先升高后降低,在Cu~(2+)浓度为200 mg·L-1时含量最高,达到1 200 mg·g-1FW; Cd~(2+)胁迫对叶绿素含量和根系活力无明显影响。根系活力在Zn~(2+)浓度为100 mg·L~(-1)时最强,随着Cu~(2+)浓度的增加而减弱。低浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)对过氧化物酶活性和可溶性糖含量都具有促进作用。Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫时对可溶性蛋白、叶绿素和丙二醛含量均无明显影响,随着复合胁迫时浓度的增加,可溶性糖含量和根系活力先增后减。这表明麻疯树对三种重金属的胁迫具有一定的抗性,过高浓度的胁迫会影响麻疯树幼苗生理生化的一些指标,但是麻疯树可以通过自身的防御系统使伤害降到最小。此外,重金属复合胁迫可以在一定程度上减轻单一胁迫对麻疯树幼苗造成的毒害作用。     

Two-dimensional gel analysis of the fate of oocyte nuclear proteins in the development of Xenopus laevis

The amphibian oocyte nucleus is thought to provide a maternal store of protein required in embryogenesis. The fate of germinal vesicle proteins has been studied by comparing polypeptide patterns of oocytes, embryos, and several adult organs of Xenopus laevis on two-dimensional gels. A combination of silver staining and fluorography of radiolabeled protein on gels was used to analyze maternal and newly synthesized polypeptides in embryogenesis. Comparison of protein patterns was facilitated and corroborated by application of monoclonal antibodies against several germinal vesicle proteins. These were characterized by immunoblotting from two-dimensional gels, and polypeptides of identical structure were recognized in oocyte nuclei, embryos, and tadpoles. The following conclusions were drawn: (1) Almost all prevalent germinal vesicle proteins can be continuously traced in embryos up to swimming tadpole stages, although their patterns of new synthesis are greatly different, some are not radiolabeled in the embryo but solely provided by the maternal store. (2) Many of the polypeptides occurring in oocyte nuclei are also found in one or several organs of the adult. (3) Tissue specificities of germinal vesicle proteins, previously detected by immunocytochemistry with monoclonal antibodies, could be confirmed by independent biochemical methods. (4) As has been previously shown by immunohistological methods, oocyte nuclear antigens are shed into the cytoplasm of the maturing egg, and are reaccumulated in the nuclei of the embryonic cells, each at a characteristic developmental stage. These shifts between intracellular compartments are not accompanied by a change of the covalent structure of the antigen.     

Xenopus cyclin D2: Cloning and expression in oocytes and during early development

Summary— We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis cyclin D2 protein. Cyclin D2 mRNA is identified as a member of the class of maternal RNAs. It is rare and stable during embryonic development at least until tadepole. In addition, a second cDNA coding for a truneated version of cyclin D2 was also isolated. Mieroinjection of cyclin D2 into oocytes undergoing meiotic maturation and parthenogenetic activation reveals that the protein is stable for several hours, independently of the ubiquitin-mediated degradation of cyclin B2 that takes place periodically during this process. Microinjected cyclin D2 localizes both in the cytoplasm and in the nucleus of oocyte. In somatic cells, it is well established that cyclin D2 is almost exclusively nuclear and very labile. The unusual behaviour of cyclin D2 upon injection into oocytes may provide indications about a possible role for this protein during meiosis and early development.     

Golgi-specific DHHC Zinc Finger Protein GODZ Mediates Membrane Ca2+ Transport

The Golgi-specific zinc finger protein GODZ (palmitoyl acyltransferase/DHHC-3) mediates the palmitoylation and post-translational modification of many protein substrates that regulate membrane-protein interactions. Here, we show that GODZ also mediates Ca²⁺ transport in expressing Xenopus laevis oocytes. Two-electrode voltage-clamp, fluorescence, and ⁴⁵Ca²⁺ isotopic uptake determinations demonstrated voltage- and concentration-dependent, saturable, and substrate-inhibitable Ca²⁺ transport in oocytes expressing GODZ cRNA but not in oocytes injected with water alone. Moreover, we show that GODZ-mediated Ca²⁺ transport is regulated by palmitoylation, as the palmitoyl acyltransferase inhibitor 2-bromopalmitate or alteration of the acyltransferase DHHC motif (GODZ-DHHS) diminished GODZ-mediated Ca²⁺ transport by ∼80%. The GODZ mutation V61R abolished Ca²⁺ transport but did not affect palmitoyl acyltransferase activity. Coexpression of GODZ-V61R with GODZ-DHHS restored GODZ-DHHS-mediated Ca²⁺ uptake to values observed with wild-type GODZ, excluding an endogenous effect of palmitoylation. Coexpression of an independent palmitoyl acyltransferase (HIP14) with the GODZ-DHHS mutant also rescued Ca²⁺ transport. HIP14 did not mediate Ca²⁺ transport when expressed alone. Immunocytochemistry studies showed that GODZ and HIP14 co-localized to the Golgi and the same post-Golgi vesicles, suggesting that heteropalmitoylation might play a physiological role in addition to a biochemical function. We conclude that GODZ encodes a Ca²⁺ transport protein in addition to its ability to palmitoylate protein substrates.