Actin gene expression in developing sea urchin embryos

We show that the synthesis of actin is regulated developmentally during early sea urchin embryogenesis and that the level of synthesis of this protein parallels the steady-state amounts of the actin messenger ribonucleic acids (RNA). An in vitro translation and RNA blotting analysis of embryo RNA from several stages of early development indicated that during the first 8 h after fertilization there was a low and relatively constant level of actin messenger RNA in the embryo. Between 8 and 13 h of development, the amount of actin messenger RNA began to increase both in the cytoplasm and on polysomes, and by 18 h the amounts of actin message per embryo had risen between approximately 10- and 25-fold in the cytoplasm and between 15- and 40-fold on polysomes. Two size classes of actin messenger RNA (2.2 and 1.8 kilobases) were identified in unfertilized eggs and in all of the developmental stages examined. The amount of each actin message class increased over a similar time interval during early development. However, the amounts of these size classes in the cytoplasm relative to each other shifted between the earliest stages examined (2 to 5 h) and the hatching blastula stage (18 h), with the ratio of the 1.8-kilobase actin messenger RNA to the 2.2-kilobase actin messenger RNA increasing almost threefold during this period.     

A presumptive developmental role for a sea urchin cyclin B splice variant

Thyroglobulin is the major secretory protein of thyroid epithelial cells. Part of thyroglobulin reaches the circulation of vertebrates by transcytosis across the epithelial wall of thyroid follicles. Clearance of thyroglobulin from the circulation occurs within the liver via internalization of thyroglobulin by macrophages. Here we have analyzed the interaction of thyroglobulin with the cell surface of J774 macrophages with the aim to identify the possible thyroglobulin-binding sites on macrophages. Binding of thyroglobulin to J774 cells was saturated at approximately 100 nM thyroglobulin with a Kd of 50 nM, and it was competed by the ligand itself. Preincubation of J774 cells with thyroglobulin resulted in downregulation of thyroglobulin-binding sites, indicating internalization of thyroglobulin and its binding proteins. By affinity chromatography, two proteins from J774 cells were identified as thyroglobulin-binding proteins with an apparent molecular mass of approximately 33 kD. Unexpectedly, both proteins were identified as histone H1 by protein sequencing. The occurrence of histone H1 at the plasma membrane was further proven by biotinylation or immunolabeling of J774 cells. The in vitro interaction between histone H1 and thyroglobulin was analyzed by surface plasmon resonance that revealed a Kd at 46 nM. In situ, histone H1 was colocalized to FITC-Tg-containing endocytic compartments of Kupffer cells, i.e., liver macrophages. We conclude that histone H1 is detectable at the cell surface of macrophages where it serves as a thyroglobulin-binding protein and mediates thyroglobulin endocytosis.     

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urichin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60 degrees C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dr?bachiensis 68% and 2.5 degrees C; S. franciscanus 51% and 3.5 degrees C; Lytechinus pictus 12% and 7.5 degrees C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06-0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.     

Promoter analysis meets pattern formation: transcriptional regulatory genes in sea urchin embryogenesis

Analyses of spatial and temporal gene control mechanisms in the sea urchin embryo have identified several important trans-regulatory factors, including some that are related to known developmental control genes of the fly and mouse. Recent advances in gene perturbation technologies, including the use of antisense oligonucleotides to target mRNAs in early-stage embryos, as well as the injection of mRNAs into zygotes to express genes ectopically, have made it possible to test the functions of such factors directly.     

Phylogenetic relationships within the genus Equus and the evolution of alpha and theta globin genes

Sequences of the alpha1, alpha2 and theta globin genes from six equid species have been determined to investigate relationships within the genus Equus. Analyses using standard phylogenetic methods, or an approach designed to account for the effects of gene conversion between the alpha genes, gave broadly similar results and show that the horses diverged from the zebra/ass ancestor approximately 2.4 million years ago and that the zebra and ass species arose in a rapid radiation approximately 0.9 million years ago. These results from the alpha genes are corroborated by theta gene data and are in contrast to mitochondrial DNA studies of the phylogeny of this genus, which suggest a more gradual set of speciation events.     

Phylogenetic analysis of anthropoid relationships

The relationships of anthropoids to other primates are currently debated, as are the relationships among early fossil anthropoids and crown anthropoids. To resolve these issues, data on 291 morphological characters were collected for 57 taxa of living and fossil primates and analyzed using PAUP and MacClade. The dental evidence provides weak support for the notion of an adapid origin for anthropoids, the cranial evidence supports the tarsier-anthropoid hypothesis, and the postcranial evidence supports a monophyletic Prosimii and a monophyletic Anthropoidea. Combining these data into a single data set produces almost universal support for a tarsier-anthropoid clade nested within omomyids. Eosimias and Afrotarsius are certainly members of this clade, and probably basal anthropoids, although the Shanghuang petrosal may not belong to Eosimias. The tree derived from the combined data set resembles the tree derived from the cranial data set rather than the larger dental data set. This may be attributable to relatively slower evolution in the cranial characters. The combined data set shows Anthropoidea to be monophyletic but the features traditionally held to be anthropoid synapomorphies are found to have evolved mosaically. Parapithecines are the sister taxon to crown anthropoids; qatraniines and oligopithecids are more distantly related sister taxa. There is support for a relationship of a Tarsius + Anthropoidea clade with either washakiines on Uintanius. These elements of tree topology remain fairly stable under different assumptions sets, but overall, tree topology is not robust. Previously divergent hypotheses regarding anthropoid relationships are attributable to the use of restricted data sets. This large data set enables the adapid-anthropoid hypothesis to be rejected, and unites Tarsius, Anthropoidea and Omomyiformes within a clade, Haplorhini. However, relationships among these three taxa cannot be convincingly resolved at present.     

Ascidian actin genes: developmental regulation of gene expression and molecular evolution

Actin is a ubiquitous protein in eukaryotic cells and plays an important role in cell structure, cell motility, and the generation of contractile force in both muscle and nonmuscle cells. Multiple genes encoding muscle or nonmuscle actins have been isolated from several species of ascidians and their expression patterns have been investigated. Sequence and expression analyses of muscle actin genes have shown that ascidians have at least two distinct isoforms of muscle actin, the larval muscle and body-wall isoforms. In the ascidian Halocynthia roretzi, two clusters of actin genes are expressed in the larval muscle cells. The HrMA2/4 cluster contains at least five actin genes and the HrMA1 cluster contains a pair of actin genes whose expression is regulated by a single bidirectional promoter. cis-Regulatory elements essential for muscle-specific expression of a larval muscle actin gene HrMA4a have been identified. The adult body-wall muscle actin is clearly distinguished from the larval muscle actin by diagnostic amino acids. The adult muscle actin genes may be useful tools to investigate the mechanisms of muscle development in ascidian adults. The evolution of chordate actin genes has been inferred by comparing the organization and sequences of actin genes and performing molecular phylogenetic analysis. The results suggest a close relationship between ascidian and vertebrate actins. The chordate ancestor seems to have evolved the "chordate-type" cytoplasmic and muscle actins before its divergence into vertebrates and urochordates. The phylogenetic analysis also suggests that the vertebrate muscle actin isoforms evolved after the separation of the vertebrates and urochordates. Muscle actin genes have been used to investigate the mechanism of muscle cell regression during the evolution of anural development. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression. Actin genes have provided a unique system to study developmental and evolutionary mechanisms in chordates.     

Detection of phospholipase Cgamma in sea urchin eggs

Studies extending over a decade have provided compelling evidence to suggest that chronic expression of proinflammatory cytokines in vivo leads to unique regulatory properties that target the cognate immune response in a way that appears to be beneficial to the host. This review focuses on the prototypic proinflammatory cytokine tumour necrosis factor alpha, because recent studies of autoimmune disease in mice and man have unraveled a novel and unexpected immunosuppressive role for this inflammatory mediator during the effector phase of the autoimmune process. So far, T lymphocytes would appear to be important cellular targets of this immunoregulatory effect.     

Localization of Rho GTPase in sea urchin eggs

OBJECTIVE: Previous studies in our laboratory showed that the platelet anti-aggregating effect exerted by insulin, mediated by a nitric oxide (NO)-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP), is lost in the insulin-resistant of obesity and obese NIDDM. It is not clear 1) whether the alterations observed in obese NIDDM patients are attributable to the obesity-related insulin resistance or to diabetes per se and 2) whether insulin-resistant states present a normal or a blunted response to NO. This study has been conducted to investigate 1) the platelet sensitivity to insulin in lean NIDDM and 2) the platelet sensitivity to an NO donor, glyceryl trinitrate (GTN), in obesity and in both lean and obese NIDDM. RESEARCH DESIGN AND METHODS: We determined 1) ADP-induced platelet aggregation and platelet cGMP content in platelet-rich plasma (PRP) obtained from 11 lean NIDDM patients, after a 3-min incubation with insulin (0, 240, 480, 960, 1,920 pmol/l) and 2) ADP-induced platelet aggregation and platelet cGMP content in PRP obtained from 9 obese subjects, 11 lean and 8 obese NIDDM patients, and 18 control subjects, after a 3-min incubation with 0, 20, 40, and 100 mumol/l GTN. RESULTS: Insulin dose-dependently decreased platelet aggregation in lean NIDDM patients (P = 0.0001): with 1,920 pmol/l of insulin, ADP ED50 was 141.5 +/- 6.4% of basal values (P = 0.0001). Furthermore, insulin increased platelet cGMP (P = 0.0001) from 7.5 +/- 0.2 to 21.1 +/- 3.7 pmol/10(9) platelets. These results were similar to those previously described in healthy subjects. GTN reduced platelet aggregation in all the groups (P = 0.0001) at all the concentrations tested (P = 0.0001), but GTN IC50 values were much higher in insulin-resistant patients: 36.3 +/- 5.0 mumol/l in healthy control subjects, 26.0 +/- 6.0 mumol/l in lean NIDDM patients (NS vs. control subjects), 123.6 +/- 24.0 mumol/l in obese subjects (P = 0.0001 vs. control subjects), and 110.1 +/- 19.2 mumol/l in obese NIDDM patients (P = 0.0001 vs. control subjects). GTN dose-dependently increased platelet cGMP in all the groups (P = 0.0001 in control subjects, lean NIDDM patients, and obese subjects; P = 0.04 in obese NIDDM patients). Values reached by obese subjects and obese NIDDM patients, however, were lower than those reached by control subjects (with 100 mumol/l of GTN, P = 0.001 and P = 0.0001, respectively). In healthy control subjects and in obese subjects, the insulin:glucose ratio, used as an indirect measure of insulin sensitivity, was positively correlated to GTN IC50 (r = 0.530, P = 0.008), further suggesting that the sensitivity to NO is reduced in the presence of insulin resistance. CONCLUSIONS: The insulin anti-aggregating effect is preserved in lean NIDDM; platelet sensitivity to GTN in preserved in lean NIDDM but is reduced in the insulin-resistant states of obesity and obese NIDDM. Resistance to nitrates, therefore, could be considered another feature of the insulin-resistance syndrome.     

Synthesis and turnover of polysomal mRNAs in sea urchin embryos

The synthesis and turnover kinetics of polysomal mRNA have been measured in sea urchin embryos. Polysomes were isolated from stages ranging between mesenchyme blastula and late gastrula Strongylocentrotus purpuratus embryos which had been exposed to exogenous 3H-guanosine. The amount of radioactivity incorporated into messenger and ribosomal RNAs was determined separately as a function of time, and the precursor pool specific activity was measured in the same embryos. Synthesis and decay rate constants were extracted from the data by a least-squares procedure. Per embryo, the rate of mRNA synthesis was calculated to be about 0.13 pg min-1, while the rate of rRNA synthesis is about 0.022 pg min-1. The newly synthesized mRNA turns over with a half-time of 5.7 hr. The data support only a single decay rate for the mRNA, but small fractions of mRNA decaying at different rates cannot be excluded. Previous studies have shown that a minor fraction of the mRNA includes the least abundant, most highly diverse set of messages ("complex class" mRNAs). To determine whether mRNAs of the complex class are synthesized and degraded at similar rates, labeled mRNA was measured in hybrids formed in mRNA excess reactions with single copy DNA. These experiments showed that complex class mRNAs represent an approximately proportional amount of the new mRNA symthesis, and turn over at the same average rate as does the bulk of the mRNA. Most of the mRNAs in the embryo polysomes are newly synthesized, rather than maternal. This statement refers both to complex class mRNAs and to prevalent mRNAs. Considering the sequence homology between embryo and oocyte mRNAs shown earlier, these results indicate that many of the same structural genes active during oogenesis are being transcribed in embryos at these stages.     

Attachment and the experience and expression of emotions in romantic relationships: A developmental perspective.

In this longitudinal study, the authors tested a developmental hypothesis derived from attachment theory and recent empirical findings. Target participants were 78 individuals who have been studied intensively from infancy into their mid-20s. When targets were 20-23 years old, the authors tested the way in which interpersonal experiences at 3 pivotal points in each target's earlier social development--infancy/early childhood, early elementary school, and adolescence--predicted the pattern of positive versus negative emotions experienced with his or her romantic partner. A double-mediation model revealed that targets classified as securely attached at 12 months old were rated as more socially competent during early elementary school by their teachers. Targets' social competence, in turn, forecasted their having more secure relationships with close friends at age 16, which in turn predicted more positive daily emotional experiences in their adult romantic relationships (both self- and partner-reported) and less negative affect in conflict resolution and collaborative tasks with their romantic partners (rated by observers). These results are discussed in terms of attachment theory and how antecedent life experiences may indirectly shape events in current relationships. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Social relationships and their developmental significance.

Social competence emerges mainly from experience in close relationships. Experience in both vertical and horizontal relationships is necessary for optimum growth. These two types of relationships serve somewhat different functions in the child's development and emerge at somewhat different times. The quality of these relationships affects the child in more or less enduring ways. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Introduction of DNA into sea urchin eggs by particle gun

We have applied the particle gun method, which was developed to introduce DNA into plant cells, to sea urchin eggs and have obtained excellent expression of the introduced DNA in the embryos. The expression can be normalized by concomitantly-introducing a reference construct. The method provides a new approach to quantitative analysis of cis-regulatory elements in sea urchin embryos.     

Molecular identification of a hyperpolarization-activated channel in sea urchin sperm

Sea urchin eggs attract sperm through chemotactic peptides, which evoke complex changes in membrane voltage and in the concentrations of cyclic AMP, cyclic GMP and Ca2+ ions The intracellular signalling pathways and their cellular targets are largely unknown. We have now cloned, from sea urchin testis, the complementary DNA encoding a channel polypeptide, SPIH. Functional expression of SPIH gives rise to weakly K+-selective hyperpolarization-activated channels, whose activity is enhanced by the direct action of cAMP. Thus, SPIH is under the dual control of voltage and cAMP. The SPIH channel, which is confined to the sperm flagellum, may be involved in the control of flagellar beating. SPIH currents exhibit all the hallmarks of hyperpolarization-activated currents (Ih), which participate in the rhythmic firing of central neurons, control pacemaking in the heart, and curtail saturation by bright light in retinal photoreceptors. Because of their sequence and functional properties, Ih channels form a class of their own within the superfamily of voltage-gated and cyclic-nucleotide-gated channels.     

The response of mouse oocytes injected with sea urchin spermatozoa

Entrainment to the 24-hour light-dark cycle is of adaptive significance to mammals. Human infants are no exception, but some postnatal care habits prevalent in developed countries can interfere with the physiological mechanisms underlying circadian synchronization. We describe the physiological mechanisms of entrainment to the light-dark cycle in fetuses and newborns, and some common parental care behaviors which subject the developing circadian system of the newborn to conflicting temporal cues. Improvements in parental care are proposed which may improve the circadian synchronization of newborns, and their parents or caregivers.