Porous poly (L-lactic acid) scaffolds are optimal substrates for internal colonization by A6 mesoangioblasts and immunocytochemical analyses.

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 optimal support for cell growth, and that the internal surfaces of PLLA polymeric sponges can be colonized by mesoangioblasts, which can be submitted for in situ confocal microscopic analyses for possible monitoring of timedependent expression of differentiation markers.

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 exposure of A6 to copper or to a hypertonic medium does neither induce HSP70 synthesis nor activation of HSF1, while a constitutive binding of constitutive heat shock element binding factor was found. Together, these data suggest that mesoangioblasts constitutively express HSP70 as an 'a priori' activation mechanism, while they maintain the ability to respond to stress stimuli.

A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.

Posttraumatic stress among young children after the death of a friend or acquaintance in a terrorist bombing.

The effects of traumatic loss on children who reported a friend or acquaintance killed in the 1995 Oklahoma City bombing of a federal office building were examined. Twenty-seven children who lost a friend or acquaintance and 27 demographically matched controls were assessed eight to ten months after the bombing. All but three of the children continued to experience posttraumatic stress symptoms. Those who lost a friend watched significantly more bombing-related television coverage than those without losses. Those who lost a friend had significantly more posttraumatic stress symptoms at the time of the assessment than those who lost an acquaintance. Parents and those working with children should be alert to the impact of loss even when it involves nonrelatives.

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. Intraembryonic and intracellular hsp location has been described; and a mechanism for achievement of thermotolerance proposed. A chaperonine role for a constitutive mitochondrial hsp56 has been suggested, as well as a role for the constitutive hsp70 in cell division. Heat shock, if preceded by 12-O-tetradecanoylphorbol-12-acetate (TPA) treatment causes apoptosis.

Mental health services for children in the first two years after the 1995 Oklahoma City terrorist bombing.

Nineteen infants and children were killed in the 1995 terrorist bombing in Oklahoma City, and many were injured. More than 200 children lost one or both parents. These casualties focused attention on children in the disaster response efforts. This paper describes the development and implementation of a school-based mental health program that provided accessible services to children affected by the bombing, with an emphasis on normalization. A clinical needs assessment of all children in the Oklahoma City public school system was carried out, and clinicians provided emergency and crisis services, counseling, and support groups.

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 molecules observed in nontreated eggs. That the constitutive hsp70 plays a role in sea urchin mitosis is indicated.

Effect of the IMPase inhibitor L690,330 on sea urchin development.

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 mm on. Concentrations lower than 3.5 mm 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.

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 urchins, is also present in the nuclear extracts and is able to bind the consensus individuated in the hsp70 i.v. gene promoter.

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

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 urchin P. 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 cytoplasms but not in the nuclei. In the embryos a higher hsp75 concentration was found in the portion facing the lumen of the cells which invaginate at gastrulation.

Effect of retinoic acid and valproate on sea urchin development.

The effect of valproate and retinoic acid on Paracentrotus lividus development and fertilization is reported. Retinoic acid at concentrations between 5 microM and 7 microM causes severe disturbance of development and at 10 microM complete embryo degeneration if added in the period from fertilization on. Concentrations up to 7 microM are uneffective if retinoic acid addition is started 16 h after fertilization. Valproate at concentrations between 1 mM and 5 mM causes severe disturbances of development if added from 5 min after fertilization on. Concentrations of 20 mM are lethal to all embryos. If the drug is added 16 h after fertilization no effect is observed up to the concentration of 10 mM. The effect of valproate is specific, because its isomere 2-en at 10 mM has very little effect on development. Both drugs affect fertilization only at higher concentrations: more than 1 microM for retinoic acid and more than 10 mM for valproate.

Activation by heat shock of hsp70 gene transcription in sea urchin embryos.

We had previously shown that P. lividus embryos subjected to heat shock are unable to synthesize proteins of the hsp70 family at a detectable level before the hatching blastula stage. We show here that this is not due to the inability to synthesize the hsp70 mRNAs, which are detectable following heat shock also in early stages, although in much lower amounts per embryo than in later stages. These mRNAs are also translated, as judged by the facts that they are present in the polysomal pellet, and that they are translatable in a cell free system. As to the question of the amount of hsp70 RNAs per nucleus, we conclude that this is also higher in later than in earlier stages. The presence of hsp70 mRNAs is already detectable after heating at 4 centigrades above 20 and their amount increases with the increase of temperature in the range between 24 degrees C and 28 degrees C.

Effect of doxorubicin and phenytoin on sea urchin development.

The effect of doxorubicin and phenytoin on Paracentrotus lividus development and fertilization is reported. Doxorubicin at concentrations between 1.4 and 1.8 microM causes severe disturbances of development and at 2.0 microM, complete embryonic degeneration, if added in the period from fertilization on. Concentrations up to 20 microM are ineffective on development if the treatment is started at 12 h after fertilization. Phenytoin at concentrations between 4.0 and 7.0 microM causes severe disturbances of development, if added from 5 min after fertilization on. Concentrations of 10 microM are lethal to all embryos. Again, if the drug is added at 12 h after fertilization, no effect is observed up to the concentration of 20 microM. Both drugs are ineffective on the fertilization process up to the concentration of 20 microM for doxorubicin and of 200 microM for phenytoin.

Characterization of a new member of the sea urchin Paracentrotus lividus hsp70 gene family and its expression.

We have sequenced a second gene of the hsp70 family derived from a genomic clone of the sea urchin, Paracentrotus lividus. The structure of this gene, named hsp70IV gene, is interrupted by one intron and differs from the previously analyzed sea urchin hsp70II gene, which contains several introns. Two open reading frames of hsp70IV gene encode a predicted protein of 639 amino acids with an M(r) of 69,672. The 5' flanking region of the gene contains a putative TATA element, three heat-shock elements made up of some arrays of the 5-bp units, NGAAN and NTTCN (N = A,C,G or T), a canonic consensus sequence for binding of the regulatory activating transcription factor (ATF), and a purine box. The 3' flanking region contains four putative polyadenylation sites located at different sites downstream from the stop codon. Using Northern blot hybridization analysis, carried out using a probe corresponding to a 3' noncoding fragment (UTR) peculiar to hsp70IV gene, we found that this gene is transcribed only under heat shock (Hs) and that the transcript can be recovered from the polysomal pellet. The hsp70IV gene may be classified as a Hs gene 70 although it contains one intron.

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.

Stress proteins by zinc ions in sea urchin embryos.

In Paracentrotus lividus embryos, treatment with zinc ions induces the synthesis of the two major stress proteins with the same molecular weight as those induced by heat shock. The developmental stages responsive to zinc ion treatment are the same as those responsive to heat shock. However, zinc treatment induces a longer lasting synthesis of the stress proteins, and, unlike heat shock, does not induce thermotolerance and does not inhibit synthesis of the bulk proteins.

Isolation and characterization of a sea urchin hsp 70 gene segment.

Three clones containing Paracentrotus lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo.

Acquisition of thermotolerance in sea urchin embryos correlates with the synthesis and age of the heat shock proteins.

Preheating at 31 degrees C induces thermotolerance in Paracentrotus lividus embryos, which therefore become able to withstand 1-h treatment at the otherwise lethal temperature of 35 degrees C, and to develop normally. The acquisition of thermotolerance is positively correlated with the amount of heat shock proteins produced during the 31 degrees C treatment. Evidence is provided that the heat shock proteins, although present in the embryo for long periods after synthesis, lose their effect on thermotolerance within 3 h of the cessation of synthesis.

Response to heat shock of different sea urchin species.

It is demonstrated that sea urchin embryos of the species Sphaerechinus granularis are able to respond to heat shock by producing heat shock proteins at the same stage as embryos of Paracentrotus lividus, i.e. after hatching. Arbacia lixula embryos are able to synthesize heat shock proteins already at the stage of 64-128 blastomeres. Embryonic survival is observed if the embryos are heated at the stages at which they can synthesize the heat shock proteins. The inhibition of the bulk protein synthesis after heating at 31 degrees C is never less than 50%.

Territorial localization of heat shock mRNA production in sea urchin gastrulae.

In situ hybridization experiments with a labeled DNA probe indicate that the ability to respond to heat shock with the production of the mRNA for the 70 kd heat shock protein is segregated into the ectodermal cells already at the gastrula stage or earlier during the embryonic development of Paracentrotus lividus.