Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine     

Foraging behaviour in tadpoles of the bronze frog<Emphasis Type="Italic">Rana temporalis</Emphasis>: Experimental evidence for the ideal free distribution

The ability of bronze frogRana temporalis tadpoles (pure or mixed parental lines) to assess the profitability of food habitats and distribute themselves accordingly was tested experimentally using a rectangular choice tank with a non-continuous input design. Food (boiled spinach) was placed at two opposite ends of the choice tank in a desired ratio (1:1, 1:2 or 1:4) to create habitat A and B. The tadpoles in Gosner stage 28–33, pre-starved for 24 h, were introduced in an open ended mesh cylinder placed in the center of the choice tank, held for 4 min (for acclimation) and then released to allow free movement and habitat selection. The number of tadpoles foraging at each habitat was recorded at 10, 15, 20, 25 and 30 min time intervals. The actual suitability,S _i (the food available in a habitat after colonization of tadpoles) of each habitat was obtained from the equationS _i =B _i−f _i (d _i) whereB _i is basic suitability (amount of food provided at each habitat before release of tadpoles),f _i is the rate of depletion of food (lowering effect) with introduction of each tadpole, andd _i is the density of tadpoles in habitati. The expected number of tadpoles at each habitat was derived from the actual suitability. With no food in the choice tank, movement of the tadpoles in the test arena was random indicating no bias towards any end of the choice tank or the procedure. In tests with a 1:1 food ratio, the observed ratio of tadpoles (11.71: 12.28) was comparable with the expected 12:12 ratio. The observed number of tadpoles in the habitats with a 1:2 food ratio was 8.71:15.29 and 7.87:16.13 for pure and mixed parental lines respectively. In both cases, the observed ratios were close to the expected values (7:17). Likewise, in experiments with a 1:4 food ratio, the observed number of tadpoles in the two habitats (10.78:37.22) did not differ significantly from the expected ratio of 7:41. In all tests, the number ofR. temporalis tadpoles matched ideally with habitat profitability (undermatching indexK ≜ 1. The study shows that tadpoles of the bronze frog exhibit an ideal free distribution while foraging regardless of whether they are siblings or non-siblings in a group, which correlates well with their group living strategy in nature.     

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole,Alytes obstetricans Laur.

Summary Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.     

Argyrophilic nuclear and nucleolar proteins of Xenopus laevis oocytes identified by gel electrophoresis

In order to identify argyrophilic proteins of nuclei and nucleoli, in particular those responsible for the ‘nucleolar Ag staining’ widely used in cytology, we have utilized oocytes of Xenopus laevis because of the abundance of ‘pure’ extrachromosomal nucleoli. Examination of oocytes by light and electron microscopy shows that the large extrachromosomal nucleoli are heavily stained with the Ag technique and that the Ag deposits are largely enriched in, if not exclusive to, the internal, fibrillar region. The same pattern of Ag staining in internal regions of nucleoli is observed in isolated nucleoli from which soluble nuclear proteins were removed by extensive washing. Argyrophilic proteins of isolated oocyte nuclei and purified nucleoli have been identified by reaction with AgNO₃ and formaldehyde on gel-electrophoretically separated polypeptides. Among nuclear proteins, the most prominent argyrophilia is associated with nucleoplasmin, a soluble MW 30000 phosphoprotein of the nuclear sap. In addition, four minor Ag-staining nuclear proteins have been observed. By contrast, the only strongly argyrophilic protein observed on gel electrophoresis of proteins of purified nucleoli is a high molecular weight component (apparent MW 195000) which is often resolved in a characteristic ‘pair’ of closely spaced polypeptide bands. The enrichment of this high molecular weight argyrophilic protein in isolated nucleoli and the corresponding absence of argyrophilic proteins of the nuclear sap, including nucleoplasmin, indicates that this protein contributes to the nucleolus-specific Ag staining observed in histological sections. The possible nature of this polypeptide of MW 195000 is discussed.     

Occurrence of the chromoplast protein ChrA correlates with a fruit-color gene in Capsicum annuum

Plant geneticists have determined that the color of ripe fruits of sweet peppers (Capsicum annuum L.) is determined by four genes: y, c₁, c₂and cl. We have compared the electrophoretic behavior of chromoplast membrane proteins of seven varieties of C. annuum which differ in these genes. ChrA was detected only in the varieties that had a y⁺genotype, and was not affected by variations in the other three genes. The identity of ChrA was verified by probing blots of SDS gels with antiserum to ChrA. The second known chromoplast-specific protein, ChrB, was found to be independent of all four genes. No proteins correlating with c₁, c₂or cl were detected in either one- or two-dimensional gels.     

The role of amphibian prey in the diet and growth of giant water bug nymphs in Japanese rice fields

Predatory insects that depend upon particular prey animals are commonly regulated by the prey animal’s abundance. Nymphs of the giant water bug Kirkaldyia (=Lethocerus) deyrolli (Heteroptera: Belostomatidae) are predators regarded as specialists in feeding on tadpoles. We studied the ontogenetic diet shift of aquatic nymphs by quantifying instar abundance and by analyzing captured prey and prey relative abundance during the period of rice irrigation in three localities. We also evaluated the contribution of major prey items (tadpoles, frogs, and Odonata nymphs) on specific growth rates of each nymphal stage in a rearing experiment. First to third-instar nymphs of K. deyrolli fed mainly on tadpoles, regardless of differences in prey availability. Nymphs of subsequent fourth and fifth instar stages shifted from tadpoles to other prey animals within each rice field. A rearing experiment demonstrated that giant water bug nymphs provided with tadpoles had greater specific growth rates at all nymphal stages, except for the final stage, than nymphs fed other prey (frogs and Odonata nymphs). The emergence of young K. deyrolli nymphs seemed to coincide with the period during which tadpoles became abundant in the rice fields. Consumption of tadpoles seems important to allow the nymph to complete its larval development in an unstable temporary habitat. An erratum to this article can be found at     

Bromeliad ostracods pass through amphibian (Scinaxax perpusillus) and mammalian guts alive

During a study about bromeliad tadpoles (Scinax perpusillus), the ability of bromeliad ostracods (genus Elpidium) to pass unharmed through the tadpole gut was documented. Seven Elpidium were found alive inside a tadpole's digestive tract. Subsequent experiments demonstrated that Scinax tadpoles frequently ingest bromeliad ostracods, eliminating them unharmed in the faeces. Another laboratory experiment demonstrated these ostracods'ability to pass through a mammalian (mouse) gut alive. The consequences of this ability in ostracod ecology and evolution is discussed. Biotic and abiotic data from the bromeliads where the ostracods and tadpoles were collected are given.     

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication

During late mitosis and early G₁, a series of proteins are assembled onto replication origins, resulting in them becoming ‘licensed’ for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.     

Inorganic Polyphosphates and Exopolyphosphatases in Cell Compartments of the Yeast Saccharomyces cerevisiae Under Inactivation of PPX1 and PPN1 Genes

Purified fractions of cytosol, vacuoles, nuclei, and mitochondria of Saccharomyces cerevisiae possessed inorganic polyphosphates with chain lengths characteristic of each individual compartment. The most part (80–90%) of the total polyphosphate level was found in the cytosol fractions. Inactivation of a PPX1 gene encoding ~40-kDa exopolyphosphatase substantially decreased exopolyphosphatase activities only in the cytosol and soluble mitochondrial fraction, the compartments where PPX1 activity was localized. This inactivation slightly increased the levels of polyphosphates in the cytosol and vacuoles and had no effect on polyphosphate chain lengths in all compartments. Exopolyphosphatase activities in all yeast compartments under study critically depended on the PPN1 gene encoding an endopolyphosphatase. In the single PPN1 mutant, a considerable decrease of exopolyphosphatase activity was observed in all the compartments under study. Inactivation of PPN1 decreased the polyphosphate level in the cytosol 1.4-fold and increased it 2- and 2.5-fold in mitochondria and vacuoles, respectively. This inactivation was accompanied by polyphosphate chain elongation. In nuclei, this mutation had no effect on polyphosphate level and chain length as compared with the parent strain CRY. In the double mutant of PPX1 and PPN1, no exopolyphosphatase activity was detected in the cytosol, nuclei, and mitochondria and further elongation of polyphosphates was observed in all compartments.     

A nuclear protein associated with cell divisions in plants

A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (M _r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.Abbreviations M _r apparent molecular mass - Da dalton - Ig immunoglobulins - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - 2-D gel two-dimensional gel electrophoresis - 2,4-D 2,4-dichlorophenoxyacetic acid     

Changing complexity of endogenous lectin activities during juvenile development of Xenopus laevis

Galactoside-binding lectin has been isolated from whole Xenopus laevis embryos and tadpoles at four development stages: st. 24–26, 32, 41 and 47. The main lectin activity at st. 24–26 is -galactoside specific, producing a 34/35.5K doublet on SDS-PAGE. Later in development, lectin activities specific for a wide range of other sugars appear concommitant with the detection of a number of new protein bands on SDS-PAGE gels. The greatest variety of new lectin activities exists at st. 32 when lectins specific for all of the main sugar families found in nature are detected. After this stage and up to st. 47 (the beginning of metamorphosis), fewer different lectin activities are again detected. The results suggest that a complex, developmentally regulated battery of different lectins are present during early Xenopus development, perhaps with stage-specific roles to play in the control of tissue morphogenesis.     

Glycoconjugate histochemistry of mucous glands in the skin of metamorphosing <Emphasis Type="Italic">Bufo viridis</Emphasis>

Mucins are the major glycoprotein secretions of mucous glands and display important functions in amphibian skin such as regulation of water homeostasis and mechanical and chemical protection. In the present study, we evaluated the glycoconjugate contents of developing mucous glands on dorsal regions of metamorphosing Bufo viridis (Amphibia: Anura) tadpoles using an alcian blue-PAS panel and lectin histochemistry. All the conical cells of mucous glands showed weak positivity for alcian blue in 0.025 M MgCl₂ at pH 5.7 but only a few cells were positive for 0.3 M MgCl₂ at the same pH. In addition, all the conical cells of mucous glands were negative for alcian blue at pH 2.5. In lectin histochemistry, conical cells reacted strongly with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and peanut agglutinin (PNA), weakly with Maackia amurensis leucoagglutinin (MAL). These results suggest that they express predominantly mannose, galactose and partially α(2→3)-linked sialic acid containing glycoconjugates. We concluded that dorsal mucous glands of metamorphosing Bufo viridis tadpoles contain at least two different conical cell types and glycoconjugate heterogeneity of mucous glands may be related with different functions of mucins.     

The tumor supressor function of STGC3 and its reduced expression in nasopharyngeal carcinoma

STGC3 is a novel candidate tumor suppressor gene that was found to be associated with nasopharyngeal carcinoma (NPC) via the cDNA cloning and RACE processes. The biological function of the STGC3 protein and its expression level in nasopharyngeal carcinoma remain unknown. This study aimed to evaluate the STGC3 protein expression level in NPC and to investigate the inhibitory function of STGC3 as a candidate tumor suppressor gene. We assessed the expression of the STGC3 protein in NPC biopsies and normal control specimens via Western blot and immunohistochemical analysis. The expression of STGC3 as induced by doxycycline (Dox) via a tetracycline (Tet)-regulated system in human nasopharyngeal carcinoma cell line CNE2 was also established, and the effect of STGC3 restoration on the biological behavior of CNE2 was observed. A reduced level of STGC3 expression (0.978 ± 0.213 versus 0.324 ± 0.185, P < 0.05) was detected in NPC versus normal nasopharyngeal tissue by Western blot assay. Immunohistochemical assays for STGC3 detected positive staining in the nuclei and cytoplasm of epithelial cells, and the positive expression rate in NPC, 8 of 21 (38%), was lower than that in normal nasopharynx samples, 16 of 22 (72%). After STGC3 expression was restored, the growth capacity and clone formation potential of CNE2 cells in soft agar were significantly suppressed, and the cell percentage in G₀/G₁ phase increased, while the percentage of cells entering the S and G₂ phases decreased. This indicates that an abnormality in STGC3 expression is associated with nasopharyngeal carcinogenesis and that it may play an important role in controlling cell growth and regulating the cell cycle.     

Expression and subcellular localization of X-ATM during early Xenopus development

ATM, the gene mutated in ataxia telangiectasia, is a protein essential for handling DNA strand breaks. We recently isolated the Xenopus homologue of ATM, X-ATM and we report here the detailed expression pattern of the protein and the mRNA during early Xenopus development. During the cleavage stages, ATM protein was concentrated in and around the nuclei of all cells with low levels of expression also detected in the cytoplasm. Following neurulation, increased protein levels were detected in the nuclei of developing somites and in the central nervous system. Areas of high protein expression correlated with areas of increased mRNA expression which was detected in the nuclei of somites and the developing lens. Received: 2 December 1999 / Accepted: 4 February 2000     

Discovery of a Novel Alveolate Pathogen Affecting Southern Leopard Frogs in Georgia: Description of the Disease and Host Effects

In April of 2006, we observed southern leopard frog (Rana sphenocephala) tadpoles in a pond in northeast Georgia that were dying from an unknown pathogen. Examination of affected specimens, as well as PCR characterization, revealed that all were infected with a novel alveolate pathogen closely related to freshwater and marine eukaryotic organisms and, to a lesser degree, to members of the genus Perkinsus. This pathogen has been documented in numerous mortality events in anuran tadpoles in the United States, although it has not yet been named nor clearly described. We subsequently conducted a systematic survey of this and four other ponds in the same area to document the extent of the pathogen and to describe the nature of infections in leopard frog tadpoles. Of 87 live tadpoles examined, 25% were infected with the alveolate pathogen, based on visual inspection of tadpole liver tissue. Affected tadpoles frequently had enlarged abdomens, swam erratically, and could be captured by hand. All organs of infected tadpoles were infiltrated but typically to a lesser extent than the liver and kidneys, which often had hundreds of thousands of the spherical, 6-μm organisms. Infected tadpoles tended to weigh more than noninfected ones, likely due to the massive organ swelling that coincided with infections. Infected tadpoles did not differ in developmental stage from noninfected tadpoles. Infection prevalence varied widely among ponds, and in one pond, we witnessed a rapid die-off of R. spenocephala tadpoles during our surveys, although we did find infected metamorphic frogs. The rapid mortality we observed as well as the vast number of organisms seen in specimens suggests that this pathogen has tremendous transmission potential, and therefore deserves further monitoring and study.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Expression and segregation of nucleoplasmin during development in Xenopus

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.