Radioiodination studies of the envelopes from Xenopus laevis eggs

To investigate the molecular basis of the observed morphological and biological characteristics of coelomic egg envelopes (CE), vitelline envelopes (VE), and fertilization envelopes (FE) of Xenopus laevis eggs, envelopes were radioiodinated under a variety of conditions: in situ, isolated and intact, or solubilized. The distribution of 125I in envelope components was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each envelope type displayed unique profiles when iodinated in the intact state. A major constituent of VE, the 41,500 molecular weight component, was not labeled in the intact state, although the corresponding component of CE was heavily labeled. After dissociation of the envelope by guanidine-HCl or sodium dodecyl sulfate, all of the components could be radioiodinated. However, when the envelopes (VE and FE) were dissolved by heating and subsequently radioiodinated by lactoperoxidase, the resulting radioactivity profile was similar to that of the intact envelopes, suggesting that in the heat-dissolved envelope, the individual components retain similar structural relations as in the intact envelope. Quantitative but not qualitative differences were found between the inner and outer aspects of VE and FE. The significance of these findings is discussed in relation to what is known about the morphological, biological, and molecular properties of the envelopes.     

Immunogenetic studies on the cell-mediated cytotoxicity in the clawed toadXenopus laevis

In order to test for further homologies between the MHC of mammals and amphibians, experiments were conducted to assess whether lower vertebrates such as anurans were able to generate killer cells after allogeneic stimulation. The generation of cytotoxic effector cells could be obtained in outbred families and clones of isogenic frogs after in vivo priming with either irradiated allogeneic lymphocytes or with an allogeneic skin graft, provided that the immune spleen cells were restimulated in vitro with the specific irradiated cells used for priming. Effector cells generated against a definedMHC haplotype could lyse targets having one of their two haplotypes in common with the stimulators. In contrast, no lysis was observed when the target cells differed from the specific stimulators by twoMHC haplotypes.The cytotoxic activity of the MLR-restimulated lymphocytes appeared to be mediated by T cells since passage of the effector spleen cells through a nylon wool column, under conditions which removedXenopus B lymphocytes, improved killing on a per cell basis. It therefore appears that the genes responsible for the highly specialized function of T killer cells have emerged early in evolution at least at the time of the emergence of the amphibians (300 million years ago) and that they were already linked to the MHC of this species. The MHC polymorphism inXenopus seems to be lower than in mammals as evidenced by the high frequency of cross-killing observations paralleled by the high frequency of MLR identical animals found in a large outbred population.     

Optimized transgenesis in Xenopus laevis/gilli isogenetic clones for immunological studies

Xenopus laevis provides a unique animal model, alternative to mouse, to study immunology. Even though, several methodologies have been developed for the generation of transgenic Xenopus, to date none have been adapted for the X. laevis/gilli (LG) isogenetic clones that are essential for immunological studies. Since LG clones are generated via gynogenesis, transgenic methods using transgene integration into the sperm nuclei are not suited. Therefore, we have tested three alternative methods for LG transgenesis: the phiC31 integrase, the Sleeping Beauty transposase, and the I-SceI meganuclease. All three techniques produced transgenic LG clones; however, the I-SceI meganuclease was most effective. It resulted in high transgenesis efficiency (35-50%), bright nonmosaic GFP expression as well as stable germline transmission with 100% of the progeny carrying the transgene. Production of transgenic LG clones will allow us to modulate immune gene expression and further strengthen X. laevis as a biomedical model.     

31P nuclear-magnetic-resonance studies on the developing embryos of Xenopus laevis.

The concentrations of nucleoside triphosphate, inorganic phosphate and the yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by 31P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5-4.5 nmol/embryo at least until the 'spontaneous movement' stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid-phosphate and protein-phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein-phosphate resonance disappears at a greater rate than the lipid-phosphate peak. The total phosphorus content of the embryo (approximately 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development. From the resonance positions of their alpha, beta and gamma phosphate groups it is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 +/- 0.2.     

Protamine polymorphism in Xenopus laevis laevis

Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.     

Genetic and experimental studies on a new pigment mutant in Xenopus laevis.

White lethal (wl) is a recessive mutation affecting the differentiation of the three types of chromatophores in Xenopus laevis and eventually leading to the death of the mutants around stage 50. Melanophores appear at st. 33 but differentiate abnormally, remaining pale grey, and do not proliferate after st. 41. The rare xanthophores present contain only a few differentiated pterinosomes, and the iridophores consist of noniridescent white dots. When the albino gene (ap) is combined with wl, melanophores do not differentiate. Reciprocal heterotopic and orthotopic trunk neural crest grafts have shown that the defect is intrinsic to the neural crest cells but is not due, in the case of melanophores, to a tyrosinase deficiency as revealed by the dopa reaction. The mode of action of the gene, the abnormal pattern, and lethality are discussed.     

Na,K-ATPase from Xenopus laevis kidney and epidermis: ouabain interaction studies

A satisfactory purification from Xenopus laevis epidermis is presented. Ki and Kd values for the ouabain-enzyme interaction have been evaluated. Both parameters appear very low, as compared with those demonstrated in other anurans. Very low digitalis-like compound level was demonstrated in Xenopus; on the contrary in Bufo, in which these substances play a defensive role, it is very high. Our results strengthen the hypothesis of a physiological action of such compounds in regulating enzyme-driven Na+/K+ exchanges.     

TGFβ相关蛋白在爪蟾早期胚胎中的免疫组织化学的研究

By the method of immunocytochemistry, using the polyclonal antibodies raised against the 1-29 N-terminal residues of TGF beta-1, we found that the protein could bind to the antibodies was present in the early embryos of Xenopus. The protein was named TGF beta-related protein. It was distributed mainly in the endoderm from blastula (stg. 7) to late neurula. In the blastula (stg. 8), the protein was localized in the vegetal hemisphere near the floor of the blastocoel [Plate I, Fig. 1]. In the early gastrula (stg. 10.5) [Plate I, Fig. 2], it was localized in the central part of the vegetal hemisphere. In late gastrula (stg. 12), it was mainly distributed around the gastrocoel [Plate I, Fig. 3], but the fluorescence in endoderm cells (ventral part beneath the gastrocoel) was stronger than in the mesoderm cells (dorsal part of the gastrocoel). In the early neurula (stg. 14), the whole endoderm displayed strong fluorescence and the part of dorsal mesoderm (presumptive somite & notochord) close to endoderm was also found to be positively stained [Plate I, Fig. 4,5], but the part close to neural plate was negative. In The late neurula (stg. 20) [Plate I, Fig. 6], it was found in the central area of yolk mass (endoderm cells). No positive stain was detected in the unfertilized egg, embryos earlier than stage and later than stage 20/21.2+ protein in early development.     

Structural studies on site-directed mutants of domain 3 of Xenopus laevis oocyte 5 S ribosomal RNA

Base substitutions have been introduced into the highly conserved sequences of loops D and E within domain 3 of Xenopus laevis oocyte 5 S rRNA. The effects of these mutations on the solution structure of this 5 S rRNA have been studied by means of probing with nucleases, and with chemical reagents under native and semi-denaturing conditions. The data obtained with these mutants support the graphic model of Xenopus oocyte 5 S rRNA proposed by Westhof et al. In particular, our results rule out the existence of long-range base-pairing interactions between loop C and either loop D or loop E. The data also confirm that loops D and E in the wild-type 5 S RNA adopt unusual secondary structures and illustrate the importance of nucleotide sequence in the formation of intrinsic local loop conformations via non-canonical base-pairs and specific base-phosphate contacts. Consistent with this conclusion is our observation that the domain 3 fragment of Xenopus oocyte 5 S rRNA adopts the same conformation as the corresponding region in the full-length 5 S rRNA.     

Flavonoids from Iryanthera laevis

Trunk wood of Iryanthera laevis Markgr. (Myristicaceae) contains 2′,4′-dihydroxy- 4,6′dimethoxydihydrochalcone, three 2′-hydroxy-4′,5′-methylenedioxyflavans with differently substituted A-rings (7-OH-6,8-diMe; 7-OH-5,8-diMe; 5-OH-7-OMe-6,8-diMe) and 1-(2′-hydroxy-4′-methoxy-5′-methylphenyl)-3(2″-hydroxy-4″,5″-methylenedioxphenyl)- propane, as well as three additional known diarylpropanes.     

Ultrastructural studies of spermatogenesis inAnthocerotophyta V. Nuclear metamorphosis and the posterior mitochondrion ofNotothylas orbicularis andPhaeoceros laevis

Summary Ultrastructural observations reveal that the spermatozoids of the hornwortsNotothylas andPhaeoceros contain two mitochondria and not one as described previously. Mitochondrial ontogeny and nuclear metamorphosis during spermiogenesis in these plants differ from all other archegoniates. The discovery that the posterior region of the coiled nucleus (when viewed from the anterior aspect) lies to the left of the anterior, in striking contrast to the dextral coiling of the nucleus of spermatozoids of other embryophytes, underlines the isolated nature of the hornworts among land plants. As the blepharoplast develops, the numerous ovoid mitochondria initially present in the nascent spermatid fuse to form a single elongated organelle which is positioned subjacent to the MLS and extends down between the nucleus and plastid. At the onset of nuclear metamorphosis, the solitary mitochondrion has separated into a larger anterior mitochondrion (AM) associated with the MLS and a much smaller posterior mitochondrion (PM) adjacent to the plastid. The PM retains its association with the plastid and both organelles migrate around the periphery of the cell as the spline MTs elongate. By contrast, in moss spermatids, where mitochondria undergo similar fusion and division, the AM is approximately the same size as the PM and the latter is never associated with the spline. As in other archegoniates, except mosses, spline elongation precedes nuclear metamorphosis in hornworts. Irregular strands of condensed chromatin compact basipetally to produce an elongated cylindrical nucleus which is narrower in its mid-region. During this process excess nucleoplasm moves rearward. It eventually overarches the inner surface of the plastid and entirely covers the PM.Abbreviations ABB anterior basal body - AM anterior mitochondrion - LS lamellar strip - MLS multilayered structure - MT microtubule - PBB posterior basal body - PM posterior mitochondrion     

Germination studies on three critically endangered endemic angiosperm species of the Kashmir Himalaya,India

The critically endangered and perennial alpine endemic angiosperms, namely, Aquilegia nivalis, Lagotis cashmeriana and Meconopsis latifolia inhabit such habitats in the Kashmir Himalaya that are characterised by short growing season and heavy snow cover for about 3–4 months during winter season. The seeds of these species under natural conditions experience a long period of pre-chilling during winter prior to their germination in following spring season. Taking cue from such a requirement, present study investigated the effect of chilling and exogenous application of growth hormones, NO₃ ⁻ and NH₄ ⁺ on total percent germination of otherwise deep-dormant seeds of these species, under alternate light/dark and continuous dark light regimes. Prolonged pre-chilling followed by treatment of seeds with different doses of GA₃ had a pronounced stimulatory effect on the total germination percentage in all the three species. In fact, highest germination percentage in A. nivalis was recorded only when pre-chilled seeds were treated with 1.5 mM GA₃ under alternate light/dark conditions. Likewise, germination of pre-chilled seeds of L. cashmeriana with ruptured seed coats was improved when treated with various concentrations of GA₃. Seed germination in M. latifolia was also favourably influenced by treatment of pre-chilled seeds with GA₃ or nitrogen applied either as NO₃ ⁻ or as NH₄ ⁺ under alternate light/dark conditions. Treatment of seeds with kinetin (6-furfuryl-aminopurine) had no significant influence on germination percentage in any of the three species. Thus, prolonged chilling of seeds followed by their treatment with GA₃ under alternate light/dark conditions are the requirements necessary for seed germination in these species.     

Germination inhibitors

The Botanical Review - The presence of germination-inhibiting substances in plants seems to be a wide-spread phenomenon. They occur in all parts of plants —in fruit pulp, fruit coats,...     

The response of bacterial spores to vacuum treatments. II. Germination and viability studies.

The viability of Bacillus megaterium spores has been determined after exposure to vacuum dehydration at temperatures between 0 and 65 °C, for periods up to 24 hr. A curvilinear relationship has been demonstrated between viability and drying temperature, with minimum viability occuring around 15 °C and increases in viability being shown above 35 °C. In contrast to vegetative bacteria, reequilibration of the dried spores to 2 × 10^?3 or 10 Torr aqueous vapor pressure, and/or subsequent exposure to oxygen had no effect on viability. Dehydration, rehydration and oxygen treatments had no effect on the time for outgrowth of the spores or on the growth rate of the resultant vegetative cells. Physical loss of spores from samples was not demonstrated during any of these treatments. Evidence has been presented for a novel type of spore activation, which occurs during vacuum dehydration at high temperatures, to an extent that is dependent upon drying time. The mechanism of this activation is unlike that of conventional heat or chemical activation but is oxygen independent and unaffected by reequilibration to 2 × 10^?3 or 10 Torr.