A technique for detecting matrix proteins in the crystalline spicule of the sea urchin embryo.

The presence of proteins associated with the CaCO3-containing biocrystals found in a wide variety of marine organisms is well established. In these organisms, including the primitive skeleton (spicule) of the sea urchin embryo, the structural and functional role of these proteins either in the biomineralization process or in control of the structural features of the biocrystals is unclear. Recently, one of the matrix proteins of the sea urchin spicule, SM 30, has been shown to contain a carbohydrate chain (the 1223 epitope) that has been implicated in the process whereby Ca2+ is deposited as CaCo3. Because an understanding of the localization of this protein, as well as other proteins found within the spicule, is central to understanding their function, we undertook to develop methods to localize spicule matrix proteins in intact spicules, using immunogold techniques and scanning electron microscopy. Gold particles indicative of this matrix glycoprotein could not be detected on the surface of spicules that had been isolated from embryo homogenates and treated with alkaline hypochlorite to remove any associated membranous material. However, when isolated spicules were etched for 2 min with dilute acetic acid (10 mM) to expose more internal regions of the crystal, SM 30 and perhaps other proteins bearing the 1223 carbohydrate epitope were detected in the calcite matrix. These results, indicating that these two antigens are widely distributed in the spicule, suggest that this technique should be applicable to any matrix protein for which antibodies are available.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

Synergism between UV-B radiation and a pathogen magnifies amphibian embryo mortality in nature.

Previous research has shown that amphibians have differential sensitivity to ultraviolet-B (UV-B) radiation. In some species, ambient levels of UV-B radiation cause embryonic mortality in nature. The detrimental effects of UV-B alone or with other agents may ultimately affect amphibians at the population level. Here, we experimentally demonstrate a synergistic effect between UV-B radiation and a pathogenic fungus in the field that increases the mortality of amphibian embryos compared with either factor alone. Studies investigating single factors for causes of amphibian egg mortality or population declines may not reveal the complex factors involved in declines.

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.

The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast.

In the amniotes, two unique layers of cells, the epiblast and the hypoblast, constitute the embryo at the blastula stage. All the tissues of the adult will derive from the epiblast, whereas hypoblast cells will form extraembryonic yolk sac endoderm. During gastrulation, the endoderm and the mesoderm of the embryo arise from the primitive streak, which is an epiblast structure through which cells enter the interior. Previous investigations by others have led to the conclusion that the avian hypoblast, when rotated with regard to the epiblast, has inductive properties that can change the fate of competent cells in the epiblast to form an ectopic embryonic axis. Thus, it has been suggested that the hypoblast normally induces the epiblast to form a primitive streak at a specific locus. In the work reported here, an attempt was made to reexamine the issue of induction. In contrast to previous reports, it was found that the rotated hypoblast of the chicken embryo does not initiate formation of an ectopic axis in the epiblast. The embryonic axis always initiates and develops according to the basic polarity of the epiblast layer. These results provoke a reinterpretation of the issues of mesoderm induction and primitive streak initiation in the avian embryo.

The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling.

Using a reverse transcription-coupled PCR, we demonstrated that both brain and spleen type cannabinoid receptor (CB1-R and CB2-R, respectively) mRNAs are expressed in the preimplantation mouse embryo. The CB1-R mRNA expression was coincident with the activation of the embryonic genome late in the two-cell stage, whereas the CB2-R mRNA was present from the one-cell through the blastocyst stages. The major psychoactive component of marijuana (-)-delta-9-tetrahydrocannabinol [(-)-THC] inhibited forskolin-stimulated cAMP generation in the blastocyst, and this inhibition was prevented by pertussis toxin. However, the inactive cannabinoid cannabidiol (CBD) failed to influence this response. These results suggest that cannabinoid receptors in the embryo are coupled to inhibitory guanine nucleotide binding proteins. Further, the oviduct and uterus exhibited the enzymatic capacity to synthesize the putative endogenous cannabinoid ligand arachidonylethanolamide (anandamide). Synthetic and natural cannabinoid agonists [WIN 55,212-2, CP 55,940, (-)-THC, and anandamide], but not CBD or arachidonic acid, arrested the development of two-cell embryos primarily between the four-cell and eight-cell stages in vitro in a dose-dependent manner. Anandamide also interfered with the development of eight-cell embryos to blastocysts in culture. The autoradiographic studies readily detected binding of [3H]anandamide in embryos at all stages of development. Positive signals were present in one-cell embryos and all blastomeres of two-cell through four-cell embryos. However, most of the binding sites in eight-cell embryos and morulae were present in the outer cells. In the blastocyst, these signals were primarily localized in the mural trophectoderm with low levels of signals in the polar trophectoderm, while little or no signals were noted in inner cell mass cells.These results establish that the preimplantation mouse embryo is a target for cannabinoid ligands. Consequently, many of the adverse effects of cannabinoids observed during pregnancy could be mediated via these cannabinoid receptors. Although the physiological significance of the cannabinoid ligand-receptor signaling in normal preimplantation embryo development is not yet clear, the regulation of embryonic cAMP and/or Ca2+ levels via this signaling pathway may be important for normal embryonic development and/or implantation.

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease amyloid, binds A beta and stimulates A beta aggregation.

NACP, a 140-amino acid presynaptic protein, is the precursor of NAC [the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease (AD) amyloid], a peptide isolated from and immunologically localized to brain amyloid of patients afflicted with AD. NACP produced in Escherichia coli bound to A beta peptides, the major component of AD amyloid. NACP bound to A beta 1-38 and A beta 25-35 immobilized on nitrocellulose but did not bind to A beta 1-28 on the filter under the same conditions. NACP binding to A beta 1-38 was abolished by addition of A beta 25-35 but not by A beta 1-28, suggesting that the hydrophobic region of the A beta peptide is critical to this binding. NACP-112, a shorter splice variant of NACP containing the NAC sequence, bound to A beta, but NACP delta, a deletion mutant of NACP lacking the NAC domain, did not bind A beta 1-38. Furthermore, binding between NACP-112 and A beta 1-38 was decreased by addition of peptide Y, a peptide that covers the last 15 residues of NAC. In an aqueous solution, A beta 1-38 aggregation was observed when NACP was also present in an incubation mixture at a ratio of 1:125 (NACP/A beta), whereas A beta 1-38 alone or NACP alone did not aggregate under the same conditions, suggesting that the formation of a complex between A beta and NACP may promote aggregation of A beta. Thus, NACP can bind A beta peptides through the specific sequence and can promote A beta aggregation, raising the possibility that NACP may play a role in the development of AD amyloid.

Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo.

In the sea urchin embryo, the lineage founder cells whose polyclonal progenies will give rise to five different territories are segregated at the sixth division. To investigate the mechanisms by which the fates of embryonic cells are first established, we looked for temporal and spatial expression of homeobox genes in the very early cleavage embryos. We report evidence that PlHbox12, a paired homeobox-containing gene, is expressed in the embryo from the 4-cell stage. The abundance of the transcripts reaches its maximum when the embryo has been divided into the five polyclonal territories--namely at the 64-cell stage--and it abruptly declines at later stages of development. Blastomere dissociation experiments indicate that maximal expression of PlHbox12 is dependent on intercellular interactions, thus suggesting that signal transduction mechanisms are responsible for its transcriptional activation in the early cleavage embryo. Spatial expression of PlHbox12 was determined by whole-mount in situ hybridization. PlHbox12 transcripts in embryos at the fourth, fifth, and sixth divisions seem to be restricted to the conditionally specified ectodermal lineages. These results suggest a possible role of the PlHbox12 gene in the early events of cell specification of the presumptive ectodermal territories.

De novo biosynthesis of the aggregation pheromone components ipsenol and ipsdienol by the pine bark beetles Ips paraconfusus Lanier and Ips pini (Say) (Coleoptera: Scolytidae).

The California five-spined ips, Ips paraconfusus Lanier, produces the myrcene-derived acyclic monoterpene alcohols ipsenol (2-methyl-6-methylene-7-octen-4-ol) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) as components of its aggregation pheromone. The pine engraver beetle, Ips pini (Say), produces only ipsdienol. Previous studies have shown that myrcene, a monoterpene in the pines colonized by these beetles, is a direct precursor to these pheromone components. In vivo radiolabeling studies reported here showed that male I. paraconfusus incorporated [1-14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol), while male I. pini incorporated [1-14C]acetate into ipsdienol and amitinol. Females of these species produced neither labeled nor unlabeled pheromone components. The purified radiolabeled monoterpene alcohols from-males were identified by comparison of their HPLC and GC retention times with those of unlabeled standards. HPLC-purified fractions containing the individual radiolabeled components were analyzed by GC-MS and were shown to include only the pure alcohols. To further confirm that ipsdienol and ipsenol were radiolabeled, diastereomeric ester derivatives of the isolated alcohols were synthesized and analyzed by HPLC and GC-MS. After derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring stereochemistry. The results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles.

Nonsteroidal antiinflammatory drugs cause apoptosis and induce cyclooxygenases in chicken embryo fibroblasts.

Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.