Syncytial perforations of human embryo membranes

An electron microscopy study of the anlage of cerebral cortex of human embryo has been carried with the aim of determining the presence of syncytial interneuronal connections in embryogenesis. It has been determined that, in part of the neurons, the glial embryo is absent and their external cell membranes are directly attached to each other by forming elongated or dotted tight junctions. Sometimes these junctions are perforated and, on their basis, the true syncytial interneuronal connections are formed. Natural structural properties of these connections are the following: formation of the base of tight membrane contacts, obligatory rounding of perforation edges, and the presence of residual particles in the form of spherical vesicles in the lumen of perforations. Results obtained allowed us to conclude that, in the anlage of cerebral cortex of embryos obtained during surgical abortion of pregnancy, apart from the formation of synaptic contacts, or until their formation, there is the possibility of syncytial interneuronal connections appearing. This should be considered during the transplantation of the developing brain.     

Substructure of membranes in cultivated chick embryo fibroblasts

Summary Electron microscope examination of the plasma membrane of chick embryo fibroblasts cultured in vitro revealed the presence of a single osmiophilic layer about 90 Å thick and a substructure composed of ovoid sub-units associated with an amorphous component. These ovoid sub-units measured approximately 112 Å along the major axis and 75 Å along the minor axis and were composed of a central core, approximately 30 Å by 60 Å, surrounded by a peripheral component.Examination of other membranous components of these cells revealed a similar ovoid subunit structure in a single layered membrane. Differences in thickness and in the sizes of ovoid sub-units were seen in these membranes. The ergastoplasmic membranes, the outer nuclear membranes, the outer mitochondrial and the Golgi membranes were found to be the thinnest.These varied in thickness from approximately 75 Å to 80 Å. The thickest membranes seen were the inner nuclear membranes. These were approximately 100 Å thick. The dimensions of the ovoid sub-units corresponded with differences in the thickness of the various membranes. These findings support the concept of a particulate substructure of cell membranes.This work was aided by Research Grant PH 5593 from the National Science Foundation. Some of the equipment used was purchased with funds from the National Institutes of Health Grant 2TI GM 326. I wish to thank Dr. Robert M. Dougherty from the Department of Microbiology who grew and supplied me with the chick embryo fibroblast cultures used in these studies, and Mrs. Ursula Feller fer her technical assistance.     

Syncytial fusion of human trophoblast depends on caspase 8

Differentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h. Trophoblast fusion and differentiation were assessed by confocal microscopy, immunohistochemistry and Western blot analysis. Culture with caspase 8 antisense oligonucleotides or peptide inhibitors reduced the fusion of cytotrophoblast with the syncytiotrophoblast, and resulted in multilayered cytotrophoblast. Caspase 8 expression was suppressed by antisense oligonucleotides and caspase 8 activities were reduced by peptide inhibitors. The organic anion-transporter hOAT-4 normally expressed in the cytotrophoblast and transferred into the syncytiotrophoblast by syncytial fusion was retained in the cytotrophoblast due to lack of fusion. We conclude that expression and activity of caspase 8 is a prerequisite for differentiation and syncytial fusion of cytotrophoblast cells.     

Molecular genetics of neuronal development in the Drosophila embryo

Making a functional nervous system involves the production of specific types of neurons in characteristic locations and their ability to find and synapse with appropriate target cells. By capitalizing on the advanced genetics and molecular biology of Drosophila, a rapidly growing number of genes have been identified that control these events. Studies of the expression and function of these genes in single, uniquely identified cells is possible because of the relative simplicity of the Drosophila embryonic nervous system. A class of neurogenic genes, including N, Dl, and E(spl), controls the emergence of the entire neuronal precursor population, whereas some of the segmentation genes, such as ftz and eve, control the fates of individual neurons. Later in development, genes encoding cell-surface molecules, called fasciclins, may be involved in the ability of growing neurons to recognize and elongate axons along specific pathways to reach their synaptic targets.     

Bicuculline-insensitive GABA binding to catfish neuronal membranes

GABA receptor binding to mammalian neuronal membranes has been classified into at least 2 subtypes—GABA_A and GABA_B binding sites. In catfish brain GABA_A receptor sites have previously been demonstrated. Evidence is now presented that under appropriate conditions which rule out GABA_A receptor binding, [³H]GABA binds to membranes prepared from catfish brain. This binding is bicuculline-insensitive but differs enough from mammalian GABA_B binding to cast some doubt on the idea that GABA_B receptors exist in catfish brain. Specific binding was detected that was saturable and exhibited a dissociation constant of 4μM. (±)Baclofen, a potent inhibitor in rat brain, was a weak inhibitor, producing a maximum of 43% inhibition. This inhibitory effect could be enhanced, however, in the presence of 320 μM isoguvacine. [³H]GABA binding was unaffected by bicuculline. Thus bicuculline-insensitive GABA binding sites exist in catfish brain but they differ in a number of ways from the GABA_B receptor site found in mammals. Furthermore, a third [³H]GABA binding site appears to exist that is both baclofen- and bicuculline-insensitive, yet is inhibited by high concentrations of isoguvacine, a known GABA_A agonist.     

Inhalational anesthetic-binding proteins in rat neuronal membranes

Molecular targets of inhaled anesthetics must be represented in the group that specifically bind these drugs, but the paucity of direct binding data has limited the number of candidates for further evaluation. To find candidate targets, we used a combination of photolabeling, two-dimensional gel electrophoresis, and mass spectrometry to identify halothane-binding targets in rat neuronal membranes. Of the 265 spots detected on the two-dimensional gels, 90 were labeled by [(14)C]halothane, and 34 were identified. Mitochondrial proteins, especially respiratory complex and voltage-dependent anion channels, dominated the labeled group, and there were several examples of subunit- and state-dependent binding. A significant correlation was found between internal protein cavities and binding in a group of proteins with high resolution structures. Therefore, in addition to identifying novel neuronal targets, these data suggest a general molecular feature, the buried cavity, as a dominant attribute of volatile anesthetic-binding sites found in a limited number of neuronal membrane proteins.     

Choline acetyltransferase-like activity bound to neuronal plasma membranes

A form of CAT-like activity was found bound present in rat brain synaptosomal membranes which could be recovered in the Triton X-114 phase. The enzyme activity was slightly activated by NaCl, had a pH maximum around 8 and showed a temperature dependence with a Q₁₀ of 2.28. It was inhibited 100% by 10^–6 M naphthyl vinyl pyridinium but not by 10^–5 M diisopropyl phosphofluoridate. The kinetics of this bound form of CAT were similar to the soluble form of the enzyme. TheK _m was 405±58 M for choline and 62±8 M for AcCoA. Five isoelectric forms were found with pH's of 4.55, 6.05, 7.06, 7.36, and 8.00 which is in contrast to the three isoelectric forms found of the soluble enzyme in rat brain. The presence of a CAT-like activity in the plasma membrane was confirmed with experiments performed using intact synaptosomes and intact cells in culture. Acetylcholine, synthesized from radioactive AcCoA by intact rat brain synaptosomes, was recovered in the incubation medium and only in the presence of exogenous choline or when the production of choline was stimulated by oleate via the activation of phospholipase D. This was also seen in experiments with intact pheochromocytoma cell cultures (PC 12) which synthesize acetylcholine that was recoverved in the incubation medium. Acetylcholine formation in the presence of choline and AcCoA was stimulated in cells that had been grown in the presence of nerve growth factor (NGF). The localization of 1% of CAT activity in a transbilayer position in the plasma membrane, could suggest a possible role of this enzymatic form in the regulation of acetylcholine synthesis.     

Presenilin 1 modifies lipid raft composition of neuronal membranes

Protein-lipid interactions in the nervous system may provide insight into the causes of neurological disorders. In this study, we elucidated if expression of human presenilin 1 (PS1) in a mouse model changes the physico-chemical properties of brain membranes. PS1 is a multifunctional transmembrane protein and part of the γ-secretase complex. This complex is critical for the production of the Alzheimer related amyloid beta peptide. Brain membranes isolated from mice expressing a human wild-type PS1 transgene are less fluid and contain higher cholesterol and sphingomyelin levels. Moreover, our data reveal significant changes in membrane micro-domains and indicate that PS1 induces the formation of lipid rafts.     

Early human embryo metabolism

Non-invasive microanalytical methods have been devised to study the energy metabolism of single human preimplantation embryos. Psyruvate, which is added routinely to all media used to culture human embryos, is consumed throughout the preimplantation period, with glucose assuming an increasing role at embryo compaction and blastocyst formation. All of the glucose consumed may be accounted for by the appearance of lactate in the incubation medium. The enzyme hexokinase my be involved in regulating this aerobic glycolysis. There is cosiderable indirect evidence for the utilisation of endogenous as opposed to exogenous energy substrates, the most likely candidate being protein. Information on early human embryo metabolismis likely to find application in a number of areas: these include the improvement of techniques for assisted human conception, notably in the selection of embryos for transfer following In Vitro Fertilisation; the diagnosis of gentic defects at the preimplantation stage; increased undersding of the causes of implantation failure and miscarriage, and the development of novel post-coital contraceptives.     

Muscle development in the grasshopper embryo. II. Syncytial origin of the extensor tibiae muscle pioneers

The extensor tibiae muscle (ETi) in the metathoracic leg of the grasshopper, which powers the jump, is among the most studied insect muscles. In contrast to many insect muscles which are simple (consisting of only a single bundle of muscle fibers), the ETi is a complex muscle which consists of an array of bundles of muscle fibers, each with a separate site of insertion on the body wall ectoderm and on the ETi apodeme ectoderm. Here we describe the embryonic development of this complex muscle. The ETi muscle develops from a single muscle pioneer (MP) which connects the initial invagination of the ETi apodeme to the wall of the femur. This MP then dramatically expands around the developing apodeme to form a large horseshoe-shaped, multinucleate cell, called the supramuscle pioneer (supra-MP); the number of nuclei in the supra-MP increases by cell fusion rather than by nuclear division. The arms of the supra-MP grow steadily longer and their outer edges begin to appear scalloped, certain areas remaining tightly apposed to the ectoderm of the wall of the leg while adjacent areas lose their adhesion and are pulled away. By about 50% of embryonic development the ETi supra-MP consists of a periodic series of bridges (cytoplasmic extensions) connecting the leg wall ectoderm with the apodeme, and linked into a giant syncytium near their inner, apodeme surface by a thin layer of cytoplasm containing hundreds of nuclei. Each bridge is surrounded by a cluster of many smaller mesoderm cells. Next the syncytium begins to divide such that by 60% the periodic bridges of the supra-MP have lost syncytial contact with each other and now themselves form an array of smaller, individual, multinucleate MPs connecting the body wall to the apodeme, each surrounded by a mass of undifferentiated mesoderm cells. This initial cycle of fusion and division is followed by a second similar cycle in which the individual mesoderm cells surrounding each MP fuse with the MP. At the same time, the MP divides into the initial bundle of smaller muscle fibers. Coincident with this division into muscle fibers is the further development of thick and thin filaments and the T-tubule system.     

Effects of dimyristoylphosphatidylethanol on the structural parameters of neuronal membranes

Fluorescent probes located in different membrane regions were used to evaluate the effects of dimyristoylphosphatidylethanol (DMPEt) on the structural parameters (transbilayer rotational and lateral mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) from the bovine cerebral cortex. An experimental procedure was used based on selective quenching of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. DMPEt increased the bulk lateral and rotational mobility, and annular lipid fluidity of SPMV lipid bilayers, and had a greater fluidizing effect on the outer monolayer than the inner monolayer. It also caused membrane proteins to cluster. These effects of DMPEt on neuronal membranes may be responsible for some, though not all, of the general anesthetic actions of ethanol.     

The brain-specific S-100 protein on neuronal cell membranes

The S-100 protein has been localized to the neuronal plasma membranes of isolated Deiters' neurons by fluorescence microscopy using fluorescein-conjugated antiserum to the protein and by immunoelectron microscopy using peroxidase-conjugated anti-S-100 antiserum. In the present study this is shown also by incubating neurons with Sepharose 4B or methylacrylate spherules to which were coupled anti-S-100 antibodies. The specificity of the antiserum is discussed in the text. The technique described can be used to study the topography of antigenic characteristics of nerve cells by using antisera insolubilized on spherules of suitable size.