PDLLA microspheres containing steroids: spray-drying, o/w and w/o/w emulsifications as preparation methods

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.     

Conservation of the 5'-terminal nucleotide sequences of ribosomal 18-S RNA in eukaryotes. Differential evolution of large and small ribosomal RNA

The 5'-terminal nucleotide sequences of ribosomal 18-S and 28-S RNA of seven species of eukaryotes including three mammals, one bird, one amphibian, one echinoderm and one slime mold, were analyzed either by means of terminal phosphorylation of RNA with polynucleotide kinase of by fingerprint analysis of uniformly labeled RNA. The following conclusions were obtained. 1. The 5'-terminal sequences of the 18-S RNA of the mouse, chicken and Dictyostelium discoideum were pUpApCp(Cp,Up)Gp---, suggesting strongly that all the eukaryotes had this same sequence at the 5'-terminus. Preliminary analysis of the 5'-termini of the 18-S RNA from human, rat, Xenopus and sea urchin cells revealed the same pUp(Np)Gp--- type 5'-terminal structure, supporting the above hypothesis. 2. The 5'-terminal sequences of the 28-S RNA of the human, rat, mouse and chicken cells were all pCpGp---, whereas those of the lower animals such as Xenopus, sea urchin and Dictyostelium were different.     

Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal membrane binding motif of the protein and resembles a putative fusion peptide. The peptide was found to mimic the behavior of its parent protein bindin with respect to (a) its high affinity for lipid bilayers, (b) the ability to aggregate and fuse vesicles, (c) the binding of Zn2+ by a histidine-rich motif, (d) the tendency to self-assemble, and (e), as indicated earlier, the adhesion to cell surface polysaccharides. Fluorescence and light scattering assays were used here to monitor peptide-induced lipid mixing, leakage, and aggregation of large unilamellar sphingomyelin/cholesterol vesicles. For these activities, B18 requires the presence of Zn2+ ions, with which it forms oligomeric complexes and assumes a partially alpha-helical conformation, as observed by circular dichroism. We conclude that aggregation and fusion involves a "trans-complex" between peptides on apposing vesicles that are connected by Zn2+ bridges.     

Density compensation functions for spiral MRI

Vaults are large ribonucleoprotein particles that have been identified in a wide range of eukaryotic organisms. Although present in thousands of copies per cell, their function remains unknown. In this report, we identify the major vault protein in sea urchins as a 107-kDa polypeptide that copurifies with microtubules and ribosomes. Although initially identified in microtubule preparations, the sea urchin major vault protein is not predominantly microtubule-associated in vivo. Rather, the sea urchin major vault protein is present throughout the cytoplasm in eggs and embryos and in the nucleus in adult somatic cells. Within the nucleus, the sea urchin major vault protein is concentrated in the region of the nucleolus and to punctate regions of the nuclear envelope. In addition, the vault protein localizes to short linear strings juxtaposed to the exterior of the nucleus and extending outward into the cytoplasm. Based on their copurification and intracellular distribution, vaults may be involved in the nucleocytoplasmic transport of ribosomes and/or mRNA.     

Hox-type and non-Hox homeobox gene sequences in genomic DNA of the sea urchin Holopneustes purpurescens

As a preliminary step in an analysis of Hox gene expression and radial body plan specification in sea urchin development, we amplified partial homeobox sequences in H. purpurescens by PCR using degenerate primers. The primers, HoxE and HoxF (Pendleton et al., 1993), spanned a highly conserved region of 82 nucleotides encompassing amino acids 21-47 of the homeodomain. Seven Hox-type homeobox sequences and two non-Hox homeobox sequences were identified. The seven Hox-type sequences were placed provisionally in Hox paralogous groups, one in paralogous group 3, three in paralogous groups 6-8 and three in paralogous groups 9 13. The non-Hox sequences had similarities with Xlox and Gbx homeobox genes.     

No effects of DC and 60-Hz AC magnetic fields on the first mitosis of two species of sea urchin embryos

The early divisions of sea urchin eggs was used as a model to study the effects of static and of 60 Hz sinusoidal magnetic fields. Two species were used (Sphaerechinus granularis and Paracentrotus lividus). Eggs were fertilized and exposed in two separate coils to the fields (up to 8 mT). Great care was taken to control the temperature of each sample. No difference was found in the time of the first division that could not be attributed to a temperature difference between samples. Comparison is made with other published data on various species.     

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.     

Isolation of cleavage furrows from eggs of regular sea urchins and identification of furrow-specific proteins

We have developed a method for the isolation of cleavage furrows from dividing sea urchin eggs, which is applicable to various sea urchin species. The new method differs from that used for isolating cleavage furrows from sand dollar Clypeaster japonicus eggs [Yonemura, S., Mabuchi, I., and Tsukita, S. (1991) J. Cell Sci. 100, 73-84] in the type and concentration of detergent included in the isolation medium, the temperature during the treatment of dividing eggs with the isolation medium, and the centrifugation conditions. The contractile ring was included in the isolated cleavage furrows, as seen on rhodamine-phalloidin staining of actin filaments. When the furrows were isolated with the isolation medium containing both NaF and beta-glycerophosphate, which are potent protein phosphatase inhibitors, the isolated furrows were found to be accompanied by the mitotic apparatus. When the isolation was carried out in the absence of both NaF and beta-glycerophosphate, cleavage furrows without the mitotic apparatus were obtained. The development of a method of isolation of cleavage furrows from regular sea urchin eggs enabled us to compare protein constituents among furrows from different sea urchin and sand dollar species. We found that 32, 36, and 51 kDa proteins were concentrated in common in the cleavage furrows isolated from eggs of the sand dollars, C. japonicus and Scaphechinus mirabilis, and the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus nudus, on two-dimensional gel electrophoreses.     

Disturbances of the secretory stage of amelogenesis in fluorosed deer teeth: a scanning electron-microscopic study

The sea urchin sperm cell is an advantageous model for studying ligand-mediated exocytosis. Sperm can be obtained in vast quantities and induced to undergo exocytosis of the acrosomal vesicle with great synchrony. During sea urchin fertilization, egg jelly (EJ) triggers the sperm acrosome reaction (AR) which is required for sperm binding and fusion with the egg. Uncertainty exists as to the exact biochemical nature of the AR inducer. The following study was performed in an attempt to clarify the nature of the inducer. EJ from individual females (Strongylocentrotus purpuratus) was analyzed on SDS-PAGE gels. Each female had a unique composition of EJ macromolecules, but all females possessed the previously described fucose sulfate polymer (FSP). Two electrophoretic isotypes of FSP were discovered; 87% of females had only one isotype and 13% had both. Both FSP isotypes bound to the REJ protein (receptor for egg jelly) purified from sperm. The two FSP isotypes had almost equal potency in inducing the AR. EJ was fractionated by DEAE chromatography in 6 M urea/4% beta-mercaptoethanol. All AR-inducing activity coeluted with FSP. FSP, purified by trypsin digestion followed by dialysis, was twice as active as the non-trypsin-digested control at inducing the AR. EJ was digested with proteinase K, boiled in detergent and beta-mercaptoethanol, and subjected to sucrose density gradient sedimentation. The FSP and AR activity had superimposable sedimentation patterns. Purified FSP had no associated peptide component. Sperm from individual males differed in the concentration dependency of purified FSP to induce the AR. The data indicate that the 138/82 kDa EJ glycoproteins, previously thought to act as AR inducers, do not appear to be involved in triggering the AR. The data are consistent with the hypothesis that FSP is the only inducer of the AR of this sea urchin species.     

Arylsulfatase exists as non-enzymatic cell surface protein in sea urchin embryos

The physiological role of arylsulfatase (Ars) and its function during development have yet to be satisfactorily defined in any species, though the proteins are widely distributed and the genes have been cloned from various organisms. Here we report the dual location of two types of Ars in sea urchin embryos. The majority of sea urchin Ars does not exhibit enzyme activity and is extracellularly distributed in aboral ectoderm cells (nonenzymatic Ars). Only a small portion has enzyme activity and is localized in lysosomal vesicles (enzymatic Ars). The elution pattern of Ars proteins processed by DEAE-cellulose or analytical gel-column chromatography reveals that although the molecular radius of enzymatic Ars differs from that of nonenzymatic Ars, they have the same charge. Furthermore, sedimentation analysis shows that purified Ars of sea urchin embryos is soluble in the absence of divalent cations but becomes insoluble in the presence of Ca2+ or Mg2+. Taken together, the present results suggest that non-enzymatic Ars is a new member of the cell surface component or extracellular matrix. It is possible that this cell surface Ars plays an important role in morphogenesis of sea urchin embryos.     

Cloning and characterization of a developmentally regulated sea urchin cDNA encoding glutamine synthetase

A 2935-bp cDNA clone encoding glutamine synthetase (GS) was isolated from a cDNA library prepared from four-blastomere Paracentrotus lividus sea urchin embryos. The sequence consists of a 75-bp 5' untranslated region (5'-UTR) followed by a 1095-bp coding region corresponding to a 365-amino-acid (aa) protein, a 1747-bp 3'-UTR and a terminal 18-bp poly(A) tail. The encoded protein shows about 66% identical residues, as compared with human and lobster class-II GS. The sequence contains the Mn(2+)-binding aa and the highly conserved aa regions observed in other GS. Northern blot analyses show that the GS mRNA is present in the sea urchin egg and is developmentally regulated in the embryo.     

Alterations in colonic mucosal vessels in patients with cirrhosis and noncirrhotic portal hypertension

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus. The protein has been named Pl-200K or Hp-200K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal-vegetal axis in sea urchin embryos are largely unknown. Nuclear beta-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that beta-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that beta-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal-vegetal axis. Our results also reveal similarities between the sea urchin animal-vegetal axis and the vertebrate dorsal-ventral axis, suggesting that these axes share a common evolutionary origin.     

DNA sequences coding for the H2B histone of Psammechinus miliaris

Starting from restriction enzyme cleavage sites of known topologies the DNA sequences coding for two thirds of the H2B histone protein, together with some 3 inch extracistronic sequences, have been determined for the sea urchin Psammechinus miliaris. This unambiguously identifies this gene and further reveals its 5 inch-3inch polarity and accurate map position in a cloned histone DNA repeat unit.     

Is outer arm dynein intermediate chain 1 multifunctional?

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.     

Alkaline phosphatase (ALP) activity in rheumatoid arthritis (RA): its clinical significance and synthesis of ALP in RA synovium

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least six additional products: two non-muscle dystrophin isoforms transcribed from promoters located in the 5'-end region of the gene and four smaller proteins transcribed from internal promoters located further downstream. Several other genes, encoding evolutionarily related proteins, have been identified. These include a structurally very similar gene in vertebrates encoding utrophin (DRP1), which is closely related to dystrophin, and a number of small and simple genes in vertebrates or invertebrates encoding proteins similar to some of the small products of the DMD gene. We have isolated a sea urchin gene showing very strong sequence and structural homology with the DMD and utrophin genes. Sequence and intron/exon structure similarities suggest that this gene is related to a precursor of both the DMD gene and the gene encoding utrophin. The sea urchin gene has the unique complex structure of the DMD gene. There is at least one, and possibly more, product(s) transcribed from internal promoters, as well as a large product of >300 kDa containing at least three of the four major domains of dystrophin. The small product seems to be evolutionarily related to Dp116, one of the small products of the human DMD gene. Partial characterization of this gene helped us to construct an evolutionary tree connecting the vertebrate dystrophin gene family with related genes in invertebrates. The constructed evolutionary tree also implies that the vertebrate small and simple structured gene encoding a Dp71-like protein, called DRP2 , evolved from the dystrophin/utrophin ancestral large and complex gene by a duplication of only a small part of the gene.