Surface Proton Conduction of Sm-Doped CeO2-δ Thin Film Preferentially Grown on Al2O3 (0001).

Sm-doped CeO2-δ (Ce0.9Sm0.1O2-δ; SDC) thin films were prepared on Al2O3 (0001) substrates by radio frequency magnetron sputtering. The prepared thin films were preferentially grown along the [111] direction, with the spacing of the (111) plane (d111) expanded by 2.6% to compensate for a lattice mismatch against the substrate. The wet-annealed SDC thin film, with the reduced d111 value, exhibited surface protonic conduction in the low-temperature region below 100 °C. The O1s photoemission spectrum exhibits H2O and OH- peaks on the SDC surface. These results indicate the presence of physisorbed water layers and the generation of protons on the SDC (111) surface with oxygen vacancies. The protons generated on the SDC surface were conducted through a physisorbed water layer by the Grotthuss mechanism.

Air pollution and neonatal deaths in São Paulo, Brazil

Air pollution has been associated with health effects on different age groups. The present study was designed to assess the impact of daily changes in air pollutants (NO2, SO2, CO, O3, and particle matter (PM10)) on total number of daily neonatal deaths (those that occur between the first and the 28th days of life) in São Paulo, from January 1998 to December 2000, since adverse outcomes such as neonatal deaths associated with air pollution in Brazil have not been evaluated before. Generalized additive Poisson regression models were used and nonparametric smooth functions (loess) were adopted to control long-term trend, temperature, humidity, and short-term trends. A linear term was used for holidays. The association between air pollutants and neonatal deaths showed a short time lag. Interquartile range increases in PM10 (23.3 æg/m ) and SO2 (9.2 æg/m ) were associated with increases of 4 percent (95 percent CI, 2-6) and 6 percent (95 percent CI, 4-8), respectively. Instead of adopting a two-pollutant model we created an index to represent PM10 and SO2 effects. For an interquartile range increase in the index an increase of 6.3 percent (95 percent CI, 6.1-6.5) in neonatal deaths was observed. These results agree with previous studies performed by our group showing the deleterious effects of air pollutants during the perinatal period. The method reported here represents an alternative approach to analyze the relationship between highly correlated pollutants and public health problems, reinforcing the idea of the synergic effects of air pollutants in public health.

Air pollution and neonatal deaths in São Paulo, Brazil.

Air pollution has been associated with health effects on different age groups. The present study was designed to assess the impact of daily changes in air pollutants (NO2, SO2, CO, O3, and particle matter (PM10)) on total number of daily neonatal deaths (those that occur between the first and the 28th days of life) in São Paulo, from January 1998 to December 2000, since adverse outcomes such as neonatal deaths associated with air pollution in Brazil have not been evaluated before. Generalized additive Poisson regression models were used and nonparametric smooth functions (loess) were adopted to control long-term trend, temperature, humidity, and short-term trends. A linear term was used for holidays. The association between air pollutants and neonatal deaths showed a short time lag. Interquartile range increases in PM10 (23.3 microg/m(3)) and SO2 (9.2 microg/m(3)) were associated with increases of 4% (95% CI, 2-6) and 6% (95% CI, 4-8), respectively. Instead of adopting a two-pollutant model we created an index to represent PM10 and SO2 effects. For an interquartile range increase in the index an increase of 6.3% (95% CI, 6.1-6.5) in neonatal deaths was observed. These results agree with previous studies performed by our group showing the deleterious effects of air pollutants during the perinatal period. The method reported here represents an alternative approach to analyze the relationship between highly correlated pollutants and public health problems, reinforcing the idea of the synergic effects of air pollutants in public health.

Design and synthesis of fluoroquinophenoxazines that interact with human telomeric G-quadruplexes and their biological effects.

In this study we have identified a new structural motif for a ligand with G-quadruplex interaction that results in biological effects associated with G-quadruplex-interactive compounds. Fluoroquinolones have been reported to possess weak telomerase inhibitory activity in addition to their better known bacterial gyrase poisoning. Starting with a fluoroquinobenzoxazine, which has modest potency in a human topoisomerase II assay, we have designed a more potent inhibitor of telomerase that has lost its topoisomerase II poisoning activity. This fluoroquinophenoxazine (FQP) interacts with G-quadruplex structures to inhibit the progression of Taq polymerase in a G-quadruplex polymerase stop assay. In addition, we demonstrate by 1H NMR studies that this compound interacts with telomeric G-quadruplex structures by external stacking to the G-tetrad with both the unimolecular fold-over and the parallel G-quadruplex structures. A photocleavage assay confirms the FQP interaction site, which is located off center of the external tetrad but within the loop region. Molecular modeling using simulated annealing was performed on the FQP-parallel G-quadruplex complex to determine the optimum FQP orientation and key molecular interactions with the telomeric G-quadruplex structure. On the basis of the results of these studies, two additional FQP analogues were synthesized, which were designed to test the importance of these key interactions. These analogues were evaluated in the Taq polymerase stop assay for G-quadruplex interaction. The data from this study and the biological evaluation of these three FQPs, using cytotoxicity and a sea urchin embryo system, were in accord with the predicted more potent telomeric G-quadruplex interactions of the initial lead compound and one of the analogues. On the basis of these structural and biological studies, the design of more potent and selective telomeric G-quadruplex-interactive compounds can be envisaged.

Cardiovascular risk factors and cardiac surgery outcomes in a multiethnic sample of men and women.

BACKGROUND: Cardiovascular disease is more prevalent in some ethnic groups than in others, as are risk factors stemming from cultural practices and values. Data on the health status of Asians and Pacific Islanders are scarce and sporadic, and data on the 2 groups are usually combined for analysis. OBJECTIVE: To determine ethnic and sex-related differences among white, Japanese, and Pacific Island subjects in cardiovascular risk factors and outcomes after coronary artery bypass graft surgery. METHODS: Data were collected from a random sample of 41 men and 19 women scheduled for nonemergent coronary artery bypass graft surgery: 19 white, 18 Japanese, and 23 Pacific Island/Hawaiian subjects. Subjects were interviewed about risk factors before surgery and were followed up for the first 20 hours after surgery. Problems that occurred during the remainder of the hospital stay were assessed by chart review. Instruments used included the Charlson Comorbidity Index, Acute Physiology and Chronic Health Evaluation II, and the Therapeutic Intervention Scoring System. RESULTS: Pacific Island and Japanese subjects differed significantly in their demographic and clinical characteristics. Pacific Islanders tended to have a more difficult postoperative course than did white subjects, whereas Japanese patients tended to have fewer problems and an easier postoperative course than other subjects. CONCLUSIONS: Further study of ethnic variations in risk factors and surgical outcomes, especially variations in comorbidities, age at the onset of signs and symptoms, and postoperative complications, is needed. Combining data obtained from Japanese and Pacific Island subjects for data analysis most likely will result in a loss of important information.

Telomere-interactive agents affect proliferation rates and induce chromosomal destabilization in sea urchin embryos.

Cationic porphyrins, which interact with guanine quadruplex (G4) telomeric folds, inhibit telomerase activity in human tumor cells. In this study, we have further examined effects of porphyrins and other telomere- and telomerase-interactive agents on proliferation rates and chromosome stability in a novel in vivo model, developing sea urchin embryos. We studied two porphyrins: (i) TMPyP4, a potent telomerase inhibitor; and (ii) TMPyP2, an isomer of TMPyP4 and an inefficient telomerase inhibitor, azidothymine (AZT), the reverse transcriptase inhibitor, antisense phosphorothioate oligonucleotide to telomerase RNA (TAG6) and a control scrambled sequence (ODN). TMPyP4, AZT and TAG6 (but not TMPyP2 or ODN) decreased the rates of cell proliferation and increased the percentage of cells trapped in mitosis. Nuclear localization of TAG6, but not of ODN, was demonstrated with 5'-fluoresceinated analogs of TAG6 and ODN. Formation of elongated chromosomes incapable of separating in anaphase, induced by TMPyP4, AZT and TAG6, closely resembled phenotypes resulting from telomerase template mutation or dominant negative TRF2 allele. Our data suggest that G4-interactive agents exert their antiproliferative effects via chromosomal destabilization and warrant their further development as valuable anticancer tools.

Localization and characterization of blastocoelic extracellular matrix antigens in early sea urchin embryos and evidence for their proteolytic modification during gastrulation.

Previously, results were presented showing a spatiotemporal expression of matrix metalloproteases consistent with a role in remodeling the blastocoelic extracellular matrix (bECM) of the gastrulating sea urchin embryo [35]. In the present work, we provide evidence suggesting that the bECM is in fact the substrate for developmentally regulated proteolysis. Monoclonal antibody (mAb) LG11C7 was generated against testicular tissue of the sea urchin, Strongylocentrotus purpuratus, and recognizes extracellular matrix antigens overlying the perivisceral epithelium. Indirect immunofluorescence microscopy shows that mAb LG11C7 cross-reacts with components of the basal lamina lining the blastocoeles of early embryos and Western immunoblots of detergent extracts indicate that it recognizes gastrula-stage antigens with M(r)s of 158, 68, and 37 kDa. Glycosidase treatments reveal that the embryonal antigens contain multiple N-linked oligosaccharides. Developmental studies employing immunoprecipitations and Western blot analyses of staged embryonal detergent extracts show that the 68-kDa antigen appears between 18 and 24 h after fertilization and is accompanied by a substantial increase in the 37-kDa antigen. Thus, the appearances of the 68- and 37-kDa antigens occur during the blastula-gastrula transition, and their spatiotemporal expression is similar to that of the matrix metalloproteases reported previously. The appearance of the 68-kDa antigen and the increase in the 37-kDa antigen may be blocked by exposing the embryos to the metalloprotease inhibitor 1,10-phenanthroline, which also blocks gastrulation reversibly. These results suggest (1) that the 68- and 37-kDa antigens are products of developmentally regulated proteolysis of a basal laminar glycoprotein, and (2) that this proteolysis is required for the cell-cell/cell-matrix interactions and morphogenetic movements associated with normal gastrulation in the sea urchin embryo.

Developmentally regulated protease expression during sea urchin embryogenesis.

A temporal study of protease expression employing the technique of SDS-PAGE gelatin substrate zymography revealed a definitive appearance of proteases during early development in the sea urchins, Lytechinus pictus and Strongylocentrotus purpuratus. The levels of these proteases increase substantially during gastrulation in each species. The two major proteases with relative molecular masses of 57 and 50 kDa were found to be inhibited by the zinc chelator, 1,10-phenanthroline, the more nonspecific metal chelator, EDTA, and the reducing agent, dithiothreitol. The serine protease inhibitor, benzamidine, exerted no effects on the activities of these proteases, and both enzymes exhibited activity in the neutral to slightly basic pH range. Treatment of embryos with actinomycin D, an inhibitor of transcription, beginning up to 9 hr after fertilization, inhibited the subsequent appearances of the two proteases 48 hr after fertilization, as well as any morphological changes associated with gastrulation. Treatments beginning 15 and 21 hr after fertilization resulted in increased levels of proteases that correlate with arrests at successively more advanced stages of gastrulation. SDS-PAGE zymographic analyses of five different embryo fractions indicated that the 57- and 50-kDa proteases are localized in the blastocoel, and blastocoelic protease activity was further confirmed microscopically by in situ zymography. Hence, the 57- and 50-kDa proteases are characterized as metalloproteases. Their expression is dependent on transcription of the embryonic genome, and their spatiotemporal appearance suggests an involvement in blastocoelic matrix remodeling during gastrulation.

A unique expression pattern for a sperm membrane protein during sea urchin spermatogenesis.

Specific mRNAs coding for a 63 kDa sperm membrane protein (63-SMP) were localised in Strongylocentrotus purpuratus testis sections using in situ hybridisation techniques. 35S-labelled antisense RNA probes transcribed from a 766 base pair fragment of the gene coding for the 63-SMP hybridised to all spermatogenic cells in the basal germinal epithelia of testicular acini, except the most peripherally located (least differentiated) spermatogonia. No hybridisation to the luminally located mature spermatozoa or somatic cells of the testis was observed. Using monoclonal antibody J17/30 and indirect immunofluorescence techniques, the 63-SMP was localised to the same subset of spermatogenic cells that contain the 63-SMP mRNA, suggesting that expression of this gene is transcriptionally controlled. In combination with previous studies on the expression of sperm histones and sperm binding, these results show that multiple, perhaps sequential, classes of gene activity contribute to the differentiation of sea urchin sperm.

A GPI-anchored sea urchin sperm membrane protein containing EGF domains is related to human uromodulin.

An Mr 63-kD sea urchin sperm flagellar membrane protein has been previously implicated as a possible receptor for egg jelly ligand(s) that trigger the sperm acrosome reaction (AR). The cDNA and deduced amino acid sequences of the 63-kD protein are presented. The open reading frame codes for a protein of 470 amino acids which contains a putative signal sequence of 25 residues. Western blots using antibodies to two synthetic peptides confirm the sequence to be that of the 63-kD protein. The mRNA is approximately 2,300 bases in length and the gene appears to be single copy. The protein is released from sperm membrane vesicles by treatment with phosphatidylinositol-specific phospholipase C, showing that it is anchored to the flagellar membrane by glycosylphosphatidyl inositol (GPI). Although we cannot demonstrate involvement of the 63-kD protein in the AR, it is of potential interest because it shares significant similarity with the developmentally expressed proteins crumbs, notch and xotch as well as human uromodulin over a region that includes two separate EGF repeats.

Seasonal changes in testicular structure and localization of a sperm surface glycoprotein during spermatogenesis in sea urchins.

Testes of the sea urchin, Strongylocentrotus purpuratus, undergo morphological changes consistent with a reproductive season that includes the winter months in the northern hemisphere (c. November to March). These changes can be observed in the basal germinal epithelia (BGE) of testicular acini as alterations in the gradients of meiosis and spermatogenesis from the peripheral (basal) to the inner (luminal) regions. Early in the season, large numbers of spermatogonia and primary spermatocytes along with large, spherical nutritive phagocytes (NPs) occupy most of the BGE. Relatively few secondary spermatocytes, spermatids, and spermatozoa are present. In the middle of the season more spermatocytes, spermatids, and spermatozoa are observed within well-defined spermatogenic columns that run between elongated NPs. Late in the season the BGE diminishes as the number of spermatogenic cells within it decreases dramatically and the lumen becomes distended with mature spermatozoa. To trace the origin and location of a 210 KD sperm surface antigen during spermatogenesis, a monoclonal antibody that recognizes this antigen, MAb J18/2, was used to probe testis sections. Indirect immunofluorescence microscopy detected the antigen throughout the BGE. Immunogold electron microscopy further localized it to the intracellular vesicles within spermatogenic cells of all stages. In mature spermatozoa the 210 KD surface antigen was detected not only on the sperm surfaces overlying the acrosomes and tails but also within the acrosome, suggesting the presence of an intracellular store of this antigen. Other than the occasional detection of antigen within the residual bodies of NPs, which contain phagocytosed spermatozoa, no antigen was detected in somatic cells of the testis, indicating that it is generated within the spermatogenic cells themselves.

Localization of wheat germ agglutinin and antibody binding sites on the plasma membranes of sea urchin sperm heads as revealed by label-fracture and fracture-flip.

Freeze-fracture electron microscopy reveals that intramembrane particles are concentrated in a band encircling the posterior portion of the acrosome of Strongylocentrotus purpuratus sperm. Two colloidal gold labeling methods, label-fracture and replica-staining fracture-flip, were employed to show that the plant lectin wheat germ agglutinin, which recognizes a 210 kDa sperm surface glycoprotein, binds to this localized band of intramembrane particles. Monoclonal antibody J18/2, which also recognizes the 210 kDa surface glycoprotein, shows this localized binding in approximately 20% of the sperm observed in this study. The majority of sperm displayed a uniform distribution of receptor sites for monoclonal antibody J18/2. Since wheat germ agglutinin and monoclonal antibody J18/2 are known to agglutinate Strongylocentrotus purpuratus sperm but not sperm of another sea urchin, Lytechinus pictus, similar determinations were made for the latter species. Lytechinus pictus sperm are not labeled with wheat germ agglutinin and are only sparsely labeled with monoclonal antibody J18/2. The acrosomal localizations of wheat germ agglutinin and monoclonal antibody J18/2 receptors in Strongylocentrotus purpuratus sperm are consistent with the involvement of the 210 kDa surface glycoprotein in an egg jelly-induced sperm acrosome reaction. Low-temperature post-embed labeling of thin sections with wheat germ agglutinin and monoclonal antibody J18/2 show concentrations of label within the acrosomal vesicle of Strongylocentrotus purpuratus sperm, suggesting the presence of an intracellular storage site for the 210 kDa glycoprotein.

Locale and level of bindin mRNA in maturing testis of the sea urchin, Strongylocentrotus purpuratus.

This first study of the onset of spermatogenesis in the sea urchin, Strongylocentrotus purpuratus, was undertaken using individuals reared in the laboratory. Spermatogenesis commences about 11-12 months after metamorphosis in these animals. Bindin message accumulates in late spermatocytes and early spermatids which lie in the luminal germinal layer. Bindin message accumulates later than does the testis-specific histone, H2b-1, suggesting that different classes of genes are sequentially activated during the differentiation of sperm. We correlate the number of bindin mRNA molecules with morphological structure and with quantitative aspects of gonad maturation including the number of nuclei and of sperm. The results suggest that the bindin mRNA concentration in total RNA from testis at different stages of maturation reflects the change in the proportion of expressing cells in the total cell population of the testis.

Localization of bindin expression during sea urchin spermatogenesis.

Expression of the bindin gene was examined in testicular cells of the sea urchin Strongylocentrotus purpuratus. In situ hybridization studies, using an 35S-labeled antisense RNA probe transcribed from a bindin cDNA, reveal that bindin mRNAs are localized in spermatogenic cells displaced towards the lumens of maturing testicular acini. Little or no hybridization is observed in spermatogenic cells displaced towards the perivisceral epithelium or in somatic cells of the testis. A similar localization of the bindin protein itself is observed using a rhodamine-conjugated polyclonal antibody against bindin, which shows a punctate immunofluorescence pattern in late spermatogenic cells. Immunogold labeling of ultrathin sections and electron microscopy reveal that this punctate immunofluorescence is an apparent result of localized deposits of bindin in intracellular vesicles. Through the terminal stages of spermatogenesis, these bindin-containing vesicles apparently fuse to form the single acrosomal vesicle of the mature spermatozoon. These results indicate 1) that bindin mRNAs are transcribed relatively late in spermatogenesis, 2) that bindin is translated soon after production of its mRNA, 3) that bindin quickly associates with intracellular vesicles during or soon after its synthesis, and 4) that these vesicles fuse to form the single acrosomal vesicle during the terminal stage of spermatogenesis.

Changing localizations of site-specific sperm surface antigens during sea urchin fertilization.

Indirect immunofluorescence studies show that monoclonal antibody (mAb) J18/2 binds site-specifically to surface antigens localized over the acrosome and tail regions of mature Strongylocentrotus purpuratus spermatozoa. Within 5 min after induction of the acrosome reaction by exposure to egg jelly or ionophore A23187, these surface antigens become detectable over the lateral region of the head so that the entire surface of the spermatozoon is labeled. Polyspermically fertilized S. purpuratus eggs fixed at varying times after insemination and exposed to mAb J18/2 reveal that these surface antigens are quickly incorporated into the egg plasma membrane and begin to disperse as early as 1.5 min after insemination. At subsequent times, they undergo further dispersal so that by 45 min they are distributed over the entire surface of the egg. These results suggest that the sperm surface components recognized by mAb J18/2 gain the ability to disperse laterally during the acrosome reaction and proceed to do so in the egg plasma membrane after fertilization.

Induction of the cortical reaction in isolated sea urchin egg cortices: effects of Ca2+ and ionophore A23187.

The cortical reaction in isolated sea urchin (Strongylocentrotus purpuratus) egg cortices has been monitored with phase-contrast video microscopy. It was confirmed that the cortical reaction is induced by exposure to Ca2+. No induction was observed after exposure to the Ca2+-ionophore A23187, although the cortices remain sensitive to a subsequent exposure to Ca2+, and the cortical reaction in unfertilized eggs suspended in cortex isolation medium remains inducible by exposure to A23187. These results imply: (1) that A23187 does not induce the cortical reaction directly; (2) that the release of intracellular Ca2+, through which A23187 induces the cortical reaction, is not from storage sites localized entirely in the cortex; and (3) that intracellular storage sites for the Ca2+ involved in the cortical reaction are also present outside the cortex.

Changing localizations of site-specific surface antigens during sea urchin spermiogenesis.

Whole mount preparations of dissociated testicular cells from the sea urchin, Strongylocentrotus purpuratus, were exposed to monoclonal antibodies (mAbs) directed against sperm surface proteins. Indirect immunofluorescence microscopy and Western immunoblot analysis show that mAb J18/29 binds to the entire surface of the mature spermatozoon and membrane proteins ranging in relative molecular masses from 25 to 340 kDa. MAb J18/2 binds to the acrosomal and tail regions of the mature spermatozoon and mainly to a 210-kDa membrane protein. MAb J17/30 binds to the midpiece and tail regions and monospecifically to a 60-kDa membrane protein. MAb J16/33 binds specifically to the sperm midpiece but does not bind to Western immunoblots of sperm membrane proteins. With the exception of J16/33, which shows a punctate binding pattern, all of these mAbs show uniform binding over the entire surface of the early spermatid. This uniform and complete surface binding is observed through all stages of spermiogenesis for mAb J18/29. By the midspermatid stage, when tail formation first begins, but before the nucleus condenses and the cytoplasm decreases in volume, localized binding patterns of mAbs J17/30 and J16/33 become evident. Localized binding of mAb J18/2 is not observed until the late spermatid stage. These results show that the sea urchin sperm surface is composed of at least four different domains and provide the first insight into differentiation of the cell surface during sea urchin spermatogenesis.

Dispersal of sperm surface antigens in the plasma membranes of polyspermically fertilized sea urchin eggs.

Polyspermically fertilized Strongylocentrotus purpuratus eggs were fixed at varying times after insemination and exposed to a monoclonal antibody (mAb J18/29) directed against a group of sperm surface antigens. Indirect immunofluorescence microscopy reveals that the sperm surface components recognized by mAb J18/29 are quickly incorporated into the egg plasma membrane and begin to disperse as early as 1.5 min after insemination. At subsequent times after insemination, they undergo further dispersal so that by 45 min they are distributed evenly over the entire surface of the egg. These results provide evidence for the free lateral mobility of sperm membrane components in the fertilized egg.

Ribonucleoside uptake and phosphorylation during fertilization and early development of the sea-urchin, Strongylocentrotus purpuratus.

Uptake of the four ribonucleosides normally present in RNA increases nearly 50-fold shortly after fertilization in eggs of the sea-urchin, Strongylocentrotus purpuratus. Uridine, adenosine and cytidine are phosphorylated (greater than 95%) to their mono-, di- and triphosphates immediately after transport into the fertilized egg. Although guanosine is transported to an extent equal to the other three ribonucleosides, less than 12% of its phosphorylated after transport. In vitro nucleoside and nucleotide kinase assays of unfertilized egg homogenates indicate that the uridine, adenosine and cytidine kinases as well as the uridylate, adenylate, cytidylate and guanylate kinases are present in the egg prior to fertilization. Substrate competition measurements indicate that adenosine phosphorylation is catalyzed by a monospecific enzyme, while uridine and cytidine phosphorylations are catalyzed by a common kinase. Guanosine kinase activity was not detectable in unfertilized egg homogenates. Between 3 h and 5 h after fertilization the phosphorylation of transported guanosine begins to increase as it enters the embryo. By 7 h after fertilization, more than 95% of the guanosine entering the embryo is phosphorylated to the mono-, di- and triphosphates. More than 80% is phosphorylated to guanosine triphosphate. The timing of increased guanosine phosphorylation correlates with a decrease in the acid-soluble GTP pools in the embryo, suggesting that increased guanosine kinase activity is a response to increased GTP demand. These results, in view of the importance of GTP in many cellular processes, imply a crucial role for guanosine kinase activation in GTP pool maintenance and cellular metabolism during early sea-urchin development.

Effects of aphidicolin on premature condensation of sperm chromosomes in fertilized sea urchin eggs.

Unfertilized Strongylocentrotus purpuratus eggs may be treated with ammonia to initiate maternal DNA replication and the maternal cell cycle. When these eggs are polyspermically fertilized 75 min after the beginning of ammonia treatment, the nuclei of the fertilizing spermatozoa undergo premature chromosome condensation (PCC) in an apparent attempt to conform to the advanced maternal cell cycle. PCC is inhibited if maternal DNA replication is blocked by exposing the eggs to aphidicolin but will proceed if this exposure begins after replication is complete. Additionally, PCC will proceed in ammonia-activated, polyspermically fertilized anucleate merogons in the continuous presence of aphidicolin. These results suggest that the direct inhibitory effect of aphidicolin may well be limited to the replication of DNA and that the unreplicated maternal nucleus itself exerts negative control over the development of chromosome-condensing conditions in the maternal cytoplasm.