Shedding of membrane vesicles containing HSP70 and FGF-2 from A6 stem cells.

Alcune vie metaboliche evolutivamente conservate nello Sviluppo

L’ UOMO E LA SCIENZA

CONSERVED CELLULAR AND MOLECULAR MECHANISMS IN DEVELOPMENT

This review discusses examples of conserved cellular and molecular mechansims in development, including the pathway of signal transduction between the photoreceptors R8 and R7 in Drosophila, which is compared to vulval induction in Caenorhabditis elegans. The Wg pathway in Drosophila is compared, first, to the Wnt pathway in dorsal mesoderm specification in Xenopus: second, to the same pathway in sea urchins; third, to the equivalent in the mom cascade of C. elegans; and finally, to parts of the equivalent pathway in Dictyostelium discoideum. The conserved expression of some hox genes in vertebrate limb buds and in the heads or tails of several invertebrate and vertebrate embryos is also il…

Sistemas de endomembranas (RE y GOLGI), sistema vacuolar y lisosomas

The Mechanism of Cleavage

Mouse A6 stem cells release active FGF-2 into extracellular space through plasma membrane vesicles

In this study, mouse mesoangioblasts were seeded onto bidimensional matrices within three-dimensional porous scaffolds of poly (L-lactic acid) (PLLA), in the presence or absence of a type I collagen coating. The cells were observed under a scanning electron microscope and tested for their adhesion, survival and proliferation. Immunolocalization of heat shock protein (Hsp) 70, an abundant and ubiquitous intracellular protein in these cells, was also performed in sectioned cell-containing scaffolds under a confocal fluorescence microscope to determine if in situ analysis of intracellular constituents was feasible. The data show that PLLA films allow direct cell adhesion and represent an optim…

Nichel,piombo e cadmio inducono risposte cellulari differenti, attivando la sintesi di differenti HSP70 in embrioni di ricci di mare

Stress response in mesangioblast stem cells

Ribonucleic Acid

Emission of trace halogens (Br, I) from a basaltic volcano: Mount Etna

Heat Shock Proteins in Sea Urchin Embryos. (heat shock proteins/sea urchin embryos)

Stress response and apoptosis in measoangioblast stem cells

Cellule staminali A6 di topo producono vescicole che contengono HSP70 e FGF-2

Espressione basale dell’HSP70 inducibile in differenti linee staminali di topo.

A method for eluting DNA in a wide range of molecular weights from agarose gels

We have developed a simple and rapid method for recovering DNAs of a wide range of molecular weights from agarose gels. A DNA-containing gel slice is placed on a Parafilm sheet in the center of a circular (positive) electrode and covered with a drop of buffer, while a linear (negative) electrode is placed on the top of the gel and driven about 1 mm into the gel itself. When a continuous current is applied, the DNA migrates into the buffer toward the circular electrode. We have obtained almost total recovery of DNAs up to 10 kb in size. Our method may also be used, under appropriate conditions, for higher molecular weight DNAs. The yield and all the biological assays performed on the DNAs ob…

Extracellular release of Hsp70 from A6 mouse stem cells

DNA amplification in sea urchin oocytes.

In situ hybridization of ribosomal RNA withParacentrotus lividus ovaries suggests that ribosomal DNA undergoes amplification in the mononucleolate oocytes of this sea urchin.

Mechanisms of Ca2+ liberation at fertilization

The mechanisms underlying the Ca2+ release at fertilization of several animal organisms are reported. Four main classical theories are described, i.e., that of Ca2+ release following simple sperm contact and a G protein stimulation; that of simple sperm contact followed by a tyrosine kinase receptor activation; that of the necessity of introduction by sperm into the egg of molecules for Ca2+ release; and that the molecule introduced into the marine eggs for Ca2+ release is the same Ca2+. Two other mechanisms for Ca2+ release are also illustrated: that of ryanodine receptor stimulation and that of NAADP formation.

Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s.

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20 h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by…

Constitutive hsp70 is essential to mitosis during early cleavage of Paracentrotus lividus embryos: The blockage of constitutive hsp70 impairs mitosis

Localization of constitutive hsp70 in eggs and early embryos of sea urchin Paracentrotus lividus is shown by means of in situ immunostaining. An accumulation of this protein is shown in the mitotic structures (asters, spindles and centrosomes). Microinjection of anti-hsp70 antibodies into eggs causes impairment of formation of mitotic structures and of cell division. This impairment goes from a complete mitotic block, to irregular mitotic apparatus formation with irregular cleavage, depending upon the antibody concentration. The localization of hsp70 after antibody microinjection is also described. Blockage of mitotic apparatus formation by nocodazole also blocks the concentration of hsp70 …

Ultrastructure

Sequence analysis of the rDNA spacer of Paracentrotus lividus and observations about pre-rRNA processing. NTS sequence of Paracentrotus lividus rDNA.

We have isolated and sequenced one intergenic region and a small part of the flanking regions (18S and 26S rRNA coding regions) of the rRNA-encoding genes (rDNA) from the sea urchin Paracentrotus lividus. This region is about 3.8 Kb long. Northern blot hybridizations and S1 mapping experiments demonstrated the presence of a partially processed 21S rRNA precursor while has the same 5' terminus as the 32S primary precursor, also in developmental stages characterized by a low rate of rRNA synthesis.

Identification of an HSF2-like factor in sea urchin embryos and its localization in primary mesenchime cells

Intracellular and extracellular Hsp70 in A6 mouse stem cells

Identification and characterization of a constitutive HSP75 in sea urchin embryos.

Abstract An antiserum against a hsp of the 70-kDa family was prepared, by means of a fusion protein, which was able to detect a constitutive 75-kDa hsc in the sea urchinP. lividus.This hsc was present both during oogenesis and at all developmental stages. A two-dimensional electrophoresis has revealed four isolectric forms of this 75-kDa hsc. The amino acid sequence of the fragment used to prepare the anti-hsp70 antibodies revealed a 43% identity with the corresponding part of sea urchin sperm receptor, and in mature eggs a brighter immunofluorescence was seen all around the cell cortex where the receptor for sea urchin sperm is localized. In oocytes the hsp75 was localized in the cytoplasm…

Hybrids

Volatile supply process of Etna volcano deduced from the volcanic gas composition.

Ectosomes containing HSP70 and FGF-2 are released from mouse A6 stem cells

Sea urchin HSF activity in vitro and in transgenic embryos.

Evidence is provided for the presence at the physiological temperature of 20 degrees C of a heat shock transcriptor factor, HSF, in the nuclei of P.lividus embryos. This HSF is able to specifically bind in vitro the heat shock element, HSE, of the promoter of the hsp70 gene i.v., as suggested by DNA-protein binding reactions and DNAse I protection assays. Upon heat-shock, at the temperature of 31 degrees C, its ability to bind the HSE units becomes much higher. The HSF activated by heat-shock drives in vivo the transcription of the beta-galactosidase reporter gene in transgenic sea urchin gastrulae. An ATF-like transcription factor, widely described in other organisms but not at all in sea …

Localization of HSP70, Cdc2, and cyclin B in sea urchin oocytes in non-stressed conditions.

In Paracentrotus lividus embryos, a Mediterranean sea urchin species, HSP70 is present in all the cells. During cell division it localizes under normal growth conditions on the centrosomes and on the whole isolated mitotic apparatus. Now, in situ hybridization, Western blot analyses, and immunohistochemistry show that the HSP70 mRNA is present in both small and large P. lividus oocytes, that all four isoforms of HSP70 can be found also in the oocytes, and that a certain amount of HSP70 localizes on asters and spindles during polar body formation. Moreover, two representative cell-cycle related proteins, cyclin B, and Cdc2, are present both in small and large oocytes, concentrating in the ge…

Hsp70 functions: inside and outside the cell

Studies on heat shock proteins in sea urchin development

Work on stress proteins in sea urchin embryos carried out over the last 20 years is reviewed and the following major results are described. Entire sea urchin embryos, if subjected to a rise in temperature at any postblastular stage undergo a wave of heat shock protein (hsp) synthesis and survive. If subjected to the same rise between fertilization and blastula formation, they are not yet able to synthesize hsp and die. Four clones coding for the major hsp, hsp70, have been isolated and sequenced; evidence for the existence of a heat shock factor has been provided, and a mechanism for the developmental regulation of hsp synthesis discussed. Intra- embryonic and intracellular hsp location has…

Voltage gradient electrophoresis of nucleic acids on agarose gels.

A very simple method is described which allows the separation of DNA molecules in a wide molecular weight range (from 0.6 to about 30 kb) in the same electrophoresis agarose gel. This is based on the achievement of a voltage gradient through a simple device consisting of a Plexiglas plate placed slantwise with respect to the gel surface plane, submerged in the electrophoretic running buffer. Further applications of our system are also described.

Stress response and apoptosis in mesoangioblasts stem cells

Studies on sea urchin oocytes. II. Synthesis of RNA during oogenesis.

Abstract Isolated oocytes of the sea urchin Paracentrotus lividus actively incorporate 3H-uridine into RNA. Labeled RNA was analysed by sucrose gradient and acrylamide gel electrophoresis following cell fractionation. Much of the radioactivity is incorporated at the nucleolar level in the form of rRNA precursors. The kinetics of maturation of these latter suggests that this occurs at a slower rate than during embryogenesis. Other non-nucleolar RNA classes are also actively labelled and retained in the nucleus for many hours. These results are confirmed by an autoradiographic investigation.

Starfish and Xenopus oocyte maturation

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by …

Stress response in mouse stem cells

A study on the effect of the inhibition of polyadenylylation on the production of giant cytoplasmic RNA in sea urchin embryos.

It is suggested that inhibition of RNA polyadenylylation in sea urchin mesenchyme blastulae causes a disturbance of the transport of all the size classes of newly synthesized RNA from the nucleus to the cytoplasm.

Hsp70 release from mesoangioblast A6 stem cells through vesicles

Sequence of a sea urchin hsp70 gene and its 5' flanking region.

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.

Hsp70 localizes differently from chaperone Hsc70 in mouse mesoangioblasts under physiological growth conditions

Mouse A6 mesoangioblasts express Hsp70 even in the absence of cellular stress. Its expression and its intracellular localization were investigated under normal growth conditions and under hyperthermic stress. Immunofluorescence assays indicated that without any stress a fraction of Hsp70 co-localized with actin microfilaments, in the cell cortex and in the contractile ring of dividing cells, while the Hsc70 chaperone did not. Hsp70 immunoprecipitation assays confirmed that a portion of Hsp70 binds actin. Immunoblot assays showed that both proteins were present in the nucleus. After heat treatment Hsp70 and actin continued to co-localize in the leading edge of A6 cells but not on microfilame…

Hsp70 function inside and outside mouse mesoangioblast stem cells

Vicende delle scienze della vita e della biomedicina

Emission of Bromine and Iodine from Mt. Etna volcano

Constraining fluxes of volcanic bromine and iodine to the atmosphere is important given the significant role these species play in ozone depletion. However, very few such measurements have been made hitherto, such that global volcanic fluxes are poorly constrained. Here we extend the data set of volcanic Br and I degassing by reporting the first measurements of bromine and iodine emissions from Mount Etna. These data were obtained using filter packs and contemporaneous ultraviolet spectroscopic SO2 flux measurements, resulting in time-averaged emission rates of 0.7 kt yr(-1) and 0.01 kt yr(-1) for Br and I, respectively, from April to October 2004, from which we estimate global Br and I flu…

A6 stem cells release vesicles containing HSP70i

Armazon celulary organelos: citoesqueleto, mitocondria, peroxisomas y nucleo

Energetic Metabolism

Hsp70 is required for optimal cell proliferation in mouse A6 mesoangioblast stem cells.

Mouse Hsp70 (70 kDa heat shock protein) is preferentially induced by heat or stress stimuli. We previously found that Hsp70 is constitutively expressed in A6 mouse mesoangioblast stem cells, but its possible role in these cells and the control of its basal transcription remained unexplored. Here we report that in the absence of stress, Ku factor is able to bind the HSE (heat shock element) consensus sequence in vitro, and in vivo it is bound to the proximal hsp70 promoter. In addition, we show that constitutive hsp70 transcription depends on the co-operative interaction of different factors such as Sp1 (specificity protein 1) and GAGA-binding protein with Ku factor, which binds the HSE cons…

Role and regulation of HSP70 in A6 mesoangioblast stem cells

Apoptosis in sea urchin embryos.

Abstract It is demonstrated by DNA electrophoresis analysis, morphological observations and TdT in situ reaction, that Paracentrotus embryos if treated with TPA plus heat undergo an apoptotic reaction. Indication is also obtained that non treated embryos undergo spontaneous apoptosis at the early pluteus stage, expecially in the districts of arms and intestine. The possible meaning of this latter observation is discussed.

Polymorphisms in the intergenic region of the sea urchin Paracentrotus lividus ribosomal DNA

Abstract Blot-hybridizations of the sea urchin Paracentrotus lividus genomic DNA with ribosomal DNA (rDNA) probes revealed individual variations in the length and in the sequence of the non-transcribed spacer (NTS) region. The number of rDNA repeat subclasses distinguishable within any individual sea urchin is usually limited (1 to 3) with respect to the widest polymorphism of the population as a whole. The heterogeneity in sequence is revealed by the presence or the absence of specific restriction sites in the spacer region. The data obtained by the intensity of the polymorphic bands indicate that different mechanisms bring about these two types of polymorphism. Preliminary data also indic…

Genes of the sea urchin embryo: An annotated list as of December 1994

The main literature regarding gene structure and expression in sea urchin embryos is schematically reported and briefly commented upon. Although the subject has expanded particularly over the last 10 years, to which the review mostly refers, some historical reference is also given. More space is reserved to the regulation of the synthesis of histones and cytoskeletal actins, where the attention of various authors has been especially present; the regulation of such a synthesis is described both at a territorial level and a temporal level during the sea urchin development.

Molecular mechanisms in developmental biology.

Some general molecular mechanisms underlying development are described. Namely: those involved in the differentiation of the R7 receptor in Drosophila embryonic retina; those involved in the determination of embryonic axes and in polar cell differentiation, in Drosophila; those involved in the determination of the AB and P cell lineage and in vulva differentiation in Caenorhabditis embryos.

“BEP” RNAs and Proteins Are Situated in the Animal Side of Sea Urchin Unfertilized Egg, Which Can Be Recognized by Female Pronuclear Localization

Microsurgery experiments demonstrate that the animal side of the unfertilized sea urchin Paracentrotus lividus egg coincides with the side of the egg pronucleus location. It is demonstrated by means of in situ hybridization and immunostaining of whole mounts of animal or vegetal halves that the previously identified bep 1 and bep4 RNAs and their proteins are located in the animal part of the unfertilized egg and much less in the vegetal part. The addition of Fabs against BEP1 and BEP4 causes exogastrulation.

Synthesis of heat-shock proteins in developing sea urchins.

Heating sea urchin embryos at 31°C greatly reduces the synthesis of the bulk proteins, whereas it highly stimulates the synthesis of some new proteins, the main ones being two closely migrating proteins of about 70,000 daltons. The production of heat-shock proteins is obtained only if the embryos are heated after hatching. Stages which produce heat-shock proteins survive heating, whereas earlier stages, not producing heat-shock proteins, do not survive. Heat-shock proteins are not produced in the presence of actinomycin D.

Stress response in mesoangioblast stem cells

Stem cells are presumed to survive various stresses, since they are recruited to areas of tissue damage and regeneration, where inflammatory cytokines and cytotoxic cells may result in severe cell injury. We explored the ability of mesoangioblasts to respond to different cell stresses such as heat, heavy metals and osmotic stress, by analyzing heat shock protein (HSP)70 synthesis as a stress indicator. We found that the A6 mesoangioblast stem cells constitutively synthesize HSP70 in a heat shock transcription factor (HSF)-independent way. However, A6 respond to heat shock and cadmium treatment by synthesizing HSP70 over the constitutive expression and this synthesis is HSF1 dependent. The e…

Analisi dell’espressione dell’HSP70 inducubile in cellule staminali ed in tessutiu adulti di topo.

EFFECT OF THE IMPase INHIBITOR L690,330 ON SEA URCHIN DEVELOPMENT

Abstract A variety of concentrations of the IMPase inhibitor L690,330 were added to sea urchin embryos. Immediate arrest of development was obtained for concentrations from 7.5 m m on. Concentrations lower than 3.5 m m permitted gastrulation but inhibited skeletogenesis and disturbed elongation along the animal–vegetal axis. The latter results are similar to those obtained by counteracting lithium effect with myoinositol, which are suggested to be due to partial relief of IMPase inhibition.

Experimental Embryology of the Sea Urchin

Shedding of vesicles from mesoangioblast A6 stem cells

Deoxyribonucleic Acid

The role of cell interactions in the control of RNA synthesis.

Hsp70 in mesoangioblast A6 stem cells.

Cortical Layer of the Egg and Physiology of Fertilization

INTRODUCTION TO MORPHOGENESIS AND RELATED PROBLEMS

Heat-Shock Proteins in Sea Urchin Embryos

The production of heat-shock proteins in sea urchin embryos is accompanied by the appearance at the polysomal level of their relative mRNAs, as shown by their translation in a cell-free system; thus suggesting that the regulation of their production occurs at a transcriptional level. The mechanism for the inhibition of the bulk protein synthesis and for its reversal on the other hand should be looked for at a posttranscriptional level, since both these phenomena occur also in the presence of actinomycin D. The heat-shock proteins produced as early as at the mesenchyme blastula stage persist within the embryo at least till the pluteus stage.

Ciclo celular, apoptosis y envejecimiento

HIGH RESOLUTION ANALYSIS WITH A MULTI-SENSOR GAS ANALYZER, AND APPLICATIONS TO ETNA, STROMBOLI AND VULCANO ISLAND (ITALY)

Synthesis of RNA in isolated nuclei of sea urchin embryos.

It is demonstrated that isolated nuclei of sea urchin embryos are able to incorporate radioactive nucleotides into RNA. Some properties of the incorporation system are described.

Cell Dissociation and Reaggregation

Mesoangioblast A6 stem cells release vesicles containing the inducible HSP70

Enzymatic Activities

INTRODUCTION TO METABOLISM

Inducible HSP70 localization in mesoangioblast A6 stem cells

Proteins

Some Other Physiological Changes That Occur at Fertilization

Regeneration in invertebrates

The mechanisms of regeneration are reviewed from a genetic, cytological and molecular biological points of view. Planarians and Hydra have been chosen and illustrated as biological examples.

Nuclear–Cytoplasmic Interactions in Early Development