Acute environmental temperature variation affects brain protein expression, anxiety and explorative behaviour in adult zebrafish.

This study investigated the effect of 4-d acute thermal treatments at 18 °C, 26 °C (control) and 34 °C on the nervous system of adult zebrafish (Danio rerio) using a multidisciplinary approach based on behavioural tests and brain proteomic analysis. The behavioural variations induced by thermal treatment were investigated using five different tests, the novel tank diving, light and dark preference, social preference, mirror biting, and Y-Maze tests, which are standard paradigms specifically tailored for zebrafish to assess their anxiety-like behaviour, boldness, social preference, aggressiveness, and explorative behaviour, respectively. Proteomic data revealed that several proteins involved in energy metabolism, messenger RNA translation, protein synthesis, folding and degradation, cytoskeleton organisation and synaptic vesiculation are regulated differently at extreme temperatures. The results showed that anxiety-like behaviours increase in zebrafish at 18 °C compared to those at 26 °C or 34 °C, whereas anxiety-related protein signalling pathways are downregulated. Moreover, treatments at both 18 °C and 34 °C affect the exploratory behaviour that appears not to be modulated by past experiences, suggesting the impairment of fish cognitive abilities. This study is the continuation of our previous work on the effect of 21-d chronic treatment at the same constant temperature level and will enable the comparison of acute and chronic treatment effects on the nervous system function in adult zebrafish.

Increase in environmental temperature affects exploratory behaviour, anxiety and social preference in Danio rerio.

The aim of this work is to investigate the effect of a temperature increase on the behaviour of adult zebrafish (Danio rerio) maintained for 21 days at 34 °C (treatment) and 26 °C (control). The temperatures chosen are within the vital range of zebrafish and correspond to temperatures that this species encounters in the natural environment. Previous results showed that the same treatment affects the brain proteome and the behaviour of adult zebrafish by producing alterations in the proteins involved in neurotransmitter release and synaptic function and impairing fish exploratory behaviour. In this study, we have investigated the performance of treated and control zebrafish during environmental exploration by using four behavioural tests (novel tank diving, light and dark preference, social preference and mirror biting) that are paradigms for assessing the state of anxiety, boldness, social preference and aggressive behaviour, respectively. The results showed that heat treatment reduces anxiety and increases the boldness of zebrafish, which spent more time in potentially dangerous areas of the tank such as the top and the uncovered bright area and at a distance from the social group, thus decreasing protection for the zebrafish. These data suggest that the increase in ambient temperature may compromise zebrafish survival rate in the natural environment.

Environmental temperature variation affects brain protein expression and cognitive abilities in adult zebrafish (Danio rerio): A proteomic and behavioural study.

Water temperature is an important environmental parameter influencing the distribution and the health of fishes and it plays a central role in ectothermic animals. The aim of this study is to determine the effects of environmental temperature on the brain proteome and the behavioural responses in zebrafish, a widely used animal model for environmental "omics" studies. Adult specimens of wild-type zebrafish were kept at 18 °C, 34 °C and 26 °C (control) for 21 days. Proteomic data revealed that several proteins involved in cytoskeletal organization, mitochondrial regulation and energy metabolism are differently regulated at the extreme temperatures. In particular, the expression of proteins associated to synapses and neurotransmitter release is down-regulated at 18 °C and 34 °C. In both thermal conditions, fish exhibited a reduced interest for the novel environment and an impairment of cognitive abilities during Y-Maze behavioural tests. The observed pathways of protein expression are possibly associated to functional alterations of the synaptic transmission that may result in cognitive functions impairment at central nervous system level as those revealed by behavioural tests. This study indicates that temperature variations can elicit biochemical changes that may affect fish health and behaviour. This combined approach provides insights into mechanisms supporting thermal acclimation and plasticity in fishes. SIGNIFICANCE: Environmental temperature variation may impact on all levels of biological life. Understanding the impact of thermal variation on the nervous system and animal behaviour is of primary importance since the results obtained can be applied from the ecological to the biomedical fields.

Gene expression profile analysis in human T lymphocytes from patients with Down Syndrome.

Down Syndrome (DS) is caused by the presence of three copies of the whole human chromosome 21 (HC21) or of a HC21 restricted region; the phenotype is likely to have originated from the altered expression of genes in the HC21. We apply the cDNA microarray method to the study of gene expression in human T lymphocytes with trisomy 21 in comparison to normal cells. Two patients with DS were investigated, along with two normal subjects as a control, all being tested in independent, duplicated cell culture experiments. The most consistent finding was the overexpression of the superoxide dismutase gene (SOD1), located on 21q, and of MHC DR beta 3 (HLA-DRB3), GABA receptor A gamma 2 (GABRG2), acetyltransferase Coenzyme, A 2 (ACAT2) and ras suppressor protein 1 (RSU1) genes. When the data were clustered according to chromosome localization, the HC21 gene set showed, on average, the highest expression in DS cells in all the experiments. Moreover, separate clustering of patients and controls was obtained when analysis was restricted to HC21 gene expression values. These findings reinforce the specific gene dosage theory for the pathogenesis of the DS phenotype, and show a consistent overexpression of the SOD1 gene on 21q.

GeneRecords: a relational database for GenBank flat file parsing and data manipulation in personal computers.

UNLABELLED: Extracting the desired data from a database entry for later analysis is a constant need in the biological sequence analysis community; GeneRecords 1.0 is a solution for GenBank biological flat file parsing, as it implements a structured representation of each feature and feature qualifier in GenBank following import in a common database managing system usable in a personal computer (Macintosh and Windows environments). This collection of related databases enables the local management of GenBank records, allowing indexing, retrieval and analysis of both information and sequences on a personal computer. AVAILABILITY: The current release, including the FileMaker Pro runtime application (built for Windows and Macintosh environments), is freely available at http://apollo11.isto.unibo.it/software/

Modulation of caspase-3 activity by zinc ions and by the cell redox state.

It is known that DNA fragmentation during apoptosis is controlled by a number of factors, a crucial step being the caspase-operated cleavage of ICAD, the DNase inhibitor. We have previously demonstrated that hydrogen peroxide-treated lymphocytes undergo apoptosis without formation of a DNA ladder; however, the use of micromolar amounts of a Zn(2+) chelator allowed DNA cleavage at internucleosomal sites. Such results were extended in the present work, thus allowing their framing into the events related to alterations in the redox state of the cell. Apoptosis in hydrogen peroxide-treated lymphocytes was found to occur with caspase-3 activation, but the enzyme activity was found to be impaired, thus affecting internucleosomal fragmentation as well as nuclear morphology. Caspase-3 activity was found to resume upon mild Zn(2+) chelation. These results provide as well an experimental model from which apoptotic events upstream and downstream of caspase-3 activity can be examined.

White cell apoptosis in platelet concentrates.

BACKGROUND: The aim of the present study was the evaluation of the apoptosis in residual white cells (WBCs) contained in platelet concentrates (PCs) and of the relationship of this apoptosis with the concentration of inflammatory cytokines in the medium and with platelet activation. STUDY DESIGN AND METHODS: Three independent methods were used to evaluated apoptosis in WBCs present in 9 PCs, either from single donors by apheresis (SD-PCs) or from pooled buffy coats (BC-PCs). All PCs were divided in two parts, one of which was irradiated. PCs were stored up to 4 days at room temperature, and samples were withdrawn daily for analysis of apoptosis, of platelet activation (surface and soluble CD62P), and of cytokine concentration (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, and tumor necrosis factor alpha). RESULTS: Apoptosis was found to occur with storage in both irradiated and nonirradiated units. Platelet activation increased with storage time and was higher in BC-PCs. The amount of released cytokines was rather variable among PC units. Only IL-8 was consistently found to increase with storage time. CONCLUSIONS: Apoptosis of residual WBCs occurred in PC units as a function of storage time. The amount and the time course of apoptosis seem to correlate with IL-8 release rather than with platelet activation or with the occurrence of febrile nonhemolytic transfusion reactions.

Nuclear matrix protein is released from apoptotic white cells during cold (1-6 degrees C) storage of concentrated red cell units and might induce antibody response in multiply transfused patients.

BACKGROUND: A previous study showed that white cells in blood units undergo apoptosis during storage. STUDY DESIGN AND METHODS: The present study attempts to show the release of nuclear matrix protein (NMP) in the supernatants of red cell units and to determine whether antibodies against nuclear components may be present in multiply transfused patients; the methods employed were enzyme-linked immunosorbent assay, flow cytometry, microscopy, immunoblotting, immunofluorescence, and confocal laser-scanning microscopy. RESULTS: NMP is released from white cells in the supernatant of packed red cell units upon cold storage (1-6 degrees C). The concentration of NMP correlates well with the degree of apoptosis, as analyzed by flow cytometry, nuclear dye staining, and DNA gel electrophoresis. Immunofluorescence also shows that white cells undergoing apoptosis (pre-G(1) peak, as seen by propidium iodide staining and flow cytometry) have an NMP content lower than control cells, which confirms an actual release of NMP. Moreover, immunoblotting analysis and immunofluorescent staining showed that, in 4 of 38 multiply transfused patients, autoantibodies against NMPs were present without any clinical or laboratory sign of autoimmune disease. One of the sera, recognizing a 64-kDa NMP, immunostained nuclear dots that were identified as coiled bodies because of their colocalization with p 80 coilin. CONCLUSION: NMP is released in the supernatant of red cell units. The results obtained from patients suggest that nuclear proteins released during apoptosis, once transfused, may induce an immune response in multiply transfused patients.

Wild-type p53-mediated down-modulation of interleukin 15 and interleukin 15 receptors in human rhabdomyosarcoma cells.

We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression.

White cell apoptosis in packed red cells.

BACKGROUND: After the removal of the buffy coat, packed red cell (RBC) transfusion units still contain white cells that may undergo apoptosis as a result of storage conditions (1-6 degrees C). The aim of the present study was the evaluation of this phenomenon in view of the possible influence it may have on febrile nonhemolytic transfusion reactions. STUDY DESIGN AND METHODS: Three independent methods (microscopy, DNA electrophoresis, and cytometry) were used to evaluate apoptosis in white cells present in 13 RBC units. Of these units, 10 had been collected into CPD/saline-adenine-glucose-mannitol and 3 into CPDA-1; each bag was split in two parts, one of which was irradiated. RBCs were stored at 1 to 6 degrees C, and samples were periodically withdrawn for study. The proliferative capacity of stored lymphocytes was evaluated after phytohemagglutinin stimulation and tritiated thymidine incorporation. RESULTS: Apoptosis was found to occur in both granulocytes and lymphocytes, starting from the first 48 to 72 hours of storage. The choice of the anticoagulant-preservative solution and the effect of irradiation did not influence the amount and the timing of the apoptotic phenomenon. Lymphocyte proliferative capacity was found to decrease sharply with storage time. CONCLUSION: Conditions of storage in RBCs induce consistent apoptosis in residual white cells. The possible clinical implications of the relationships between apoptosis and the induction of biologic response modifiers (that may cause interleukin-mediated febrile non-hemolytic transfusion reactions) and between apoptosis and immune reactions remain to be elucidated.

Production of stem cell factor and expression of c-kit in human rhabdomyosarcoma cells: lack of autocrine growth modulation.

Human rhabdomyosarcoma cells produce autocrine and paracrine growth factors that can sustain their growth and malignancy. Here we report constitutive production of stem cell factor (SCF) by 5 of 5 human rhabdomyosarcoma cell lines both of alveolar and embryonal histotype. SCF production, ranging from 30 to 162 pg/ml, was independent from the degree of myogenic differentiation and was not modulated by exogenous addition of retinoic acid (RA) or tumor necrosis factor-alpha (TNF-alpha). Four of 5 rhabdomyosarcoma cell lines expressed the mRNA for SCF receptor c-kit, while the 5th cell line became weakly positive for c-kit mRNA only after stimulation with retinoic acid. On the cell surface, c-kit protein was detectable at very low levels in only 1 of 5 rhabdomyosarcoma cell lines and was not up-regulated by RA or TNF-alpha. Addition of anti-c-kit and anti-SCF blocking antibodies, or of exogenous SCF did not alter the in vitro growth ability of rhabdomyosarcoma cells. In conclusion, our data show that rhabdomyosarcoma cells produce consistent amounts of SCF but did not demonstrate autocrine growth modulation. SCF secretion may thus have a paracrine, rather than an autocrine activity in this tumor.

Expression of functional CD40 on human osteosarcoma and Ewing's sarcoma cells.

CD40, a membrane glycoprotein of the tumor necrosis factor receptor family, is expressed by several tumor types, including B-cell lymphomas, carcinomas, and melanoma, but little is known concerning its expression by sarcoma. We used flow cytometry to analyze the expression of CD40 in human cell lines derived from 12 osteosarcomas, 6 Ewing's sarcomas, and 5 rhabdomyosarcomas. Detectable CD40 levels ranging from low to very high were found in one-third of osteosarcomas, whereas five of six Ewing's sarcomas expressed intermediate levels of CD40; all rhabdomyosarcomas were CD40-negative. At the tissue level, two of eight primary high-grade osteosarcomas showed CD40-positive immunostaining. Osteosarcoma cells and Ewing's sarcoma cells expressing CD40 were treated with recombinant soluble CD40 ligand to analyze CD40 function. Treatment with soluble CD40 ligand increased the level of apoptotic cells and stimulated the transcription of matrix metalloproteinase 9 gene, enhancing matrix metalloproteinase 9 enzyme secretion. The results indicate that in human osteosarcoma and Ewing's sarcoma, CD40 is a functional receptor whose engagement can have opposite effects on tumor cell survival and malignancy.

Spectrophotometric determination of thiols in human lymphocytes.

A spectroscopic method for thiol analysis, based on the complexation reaction with Pd(II), is described. The proposed method is simple and sensitive and can be used for a rapid analysis of thiols in human lymphocytes.

Inhibition of lung colonisation of a mouse mammary carcinoma by therapeutic vaccination with interferon-alpha gene-transduced tumor cells.

A spontaneously metastatic murine mammary adenocarcinoma, TSA, has been transduced with the gene for interferon alpha1 (IFN-alpha). Transfectants were used for the immunotherapy of mice bearing lung colonies induced by the intravenous inoculation of non-transduced parental cells. A significant reduction in the number of tumor colonies was obtained when repeated subcutaneous administrations of mitomycin C-blocked transfectant cells were given, commencing 3 days after an intravenous challenge with TSA cells. Intraperitoneal vaccination induced a stronger anti-tumor response than subcutaneous vaccination, and the proportion of tumor-free mice reached 50%. The potency of IFN-alpha transfectants was similar to that of IFN-gamma transfectants previously obtained from TSA. Admixture of IFN-alpha and IFN-gamma transfectant cells in the same vaccine did not increase the curative effect over that of single vaccines. In nude mice vaccination with IFN-alpha or IFN-gamma transfectants did not lead to a reduction in the number of lung colonies, indicating that an intact T cell response was required for the therapeutic effect observed in immunocompetent mice.

The immune response elicited by mammary adenocarcinoma cells transduced with interferon-gamma and cytosine deaminase genes cures lung metastases by parental cells.

The parental cells of the TSA murine mammary adenocarcinoma (TSA-pc) were transfected with both the interferon-gamma (IFN-y) gene and the cytosine deaminase (CD) suicide gene to obtain a therapeutic vaccine active against TSA-pc lung metastases. Even in the absence of treatment with the prodrug 5-fluorocytosine (5-FC), the local growth of double transfectants (CD-y clones) was inhibited by a marked recruitment of granulocytes and macrophages. In mice harboring TSA-pc micrometastases, therapeutic vaccination with either IFN-gamma or CD single transfectants reduced the number of lung nodules, whereas CD-gamma double transfectants abrogated metastasis growth in up to 80% of mice. Treatment of mice with 5-FC did not alter the curative efficacy of CD-gamma double-transfectant cells. By contrast, in mice vaccinated with CD single-transfectant cells, 5-FC treatment caused a significant loss of their curative activity. Host T cells played an active role in the cure of lung metastases, because vaccination of nude mice with CD-gamma cells was uneffective.

Expression of interleukin 15 (IL-15) in human rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma.

Interleukin 15 (IL-15) is a recently discovered cytokine that stimulates lymphocyte proliferation and migration via a trimeric receptor sharing the beta and gamma signal transducing chains with the IL-2 receptor. IL-15 is typically produced by normal cells that do not release IL-2, but little information is currently available on human tumors. To assess whether human musculo-skeletal sarcomas produce IL-15, we analyzed surgical specimens and cell lines obtained from rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma. IL-15 mRNA was present in 9/9 surgical specimens (3 Ewing's sarcomas, 5 osteosarcomas and 1 rhabdomyosarcoma). The analysis of a panel of cell lines (7 derived from Ewing's sarcoma, 12 from osteosarcoma and 5 from rhabdomyosarcoma) showed that all rhabdomyosarcoma and osteosarcoma cell lines expressed IL-15 mRNA at levels ranging from low to high, while Ewing's sarcoma cells contained little or no IL-15 message. ELISA assays showed IL-15 release in a subset of rhabdomyosarcomas and osteosarcomas, but not in Ewing's sarcoma. The highest production of IL-15, in the picogram/ml range, was found in rhabdomyosarcoma cell lines RH30 and RD.

Therapy of murine mammary carcinoma metastasis with interferon gamma and MHC gene-transduced tumour cells.

Gene-transfected tumour cells were used to cure mice bearing lung metastases by the parental, non-transduced mammary adenocarcinoma (TSA-pc). Repeated subcutaneous (s.c.) administrations of mitomycin C (MitC)-treated interferon gamma (IFN-gamma) transfectants induced a 90% inhibition in the number of lung metastases. Therapeutic effect required an intact T-cell response, as shown by the lack of efficacy in nude mice. Autocrine stimulation by IFN-gamma induces specific modifications in the phenotype of transfectants that acquire a high metastatic ability and show a high expression of IFN-responsive genes; these two features were exploited to design two experimental protocols to obtain an improvement of the therapeutic effect. The increased metastatic ability of IFN-gamma transfectants was used to deliver IFN-gamma selectively to the lungs of mice bearing TSA-pc pulmonary metastases. A significant therapeutic effect was obtained when TSA-pc experimental metastases were treated by repeated intravenous (i.v.) injections of MitC IFN-gamma transfectants. Since i.v. administrations of IFN-gamma transfectants did not induce immune memory, the therapeutical effect appeared to depend on the inflammatory-like response activated by local IFN release. To exploit the autocrine stimulation of IFN-sensitive genes an IFN-gamma transfectant clone was subjected to a second transfection with an allogeneic class I MHC gene (H-2K(b) or H-2D(h)). IFN-gamma plus MHC double transfectants maintained IFN-gamma release, showed a very high expression of the MHC gene products, stimulated both macrophages and T cells, and were less tumorigenic in immunocompetent mice than the parent IFN-gamma clone. Therapeutic efficacy of double transfectant IFN-gamma plus H-2D(b) cells against TSA-pc was superior to single transfectants, showing that the reaction elicited by genetically engineered cells can be selectively tuned to increase therapeutic efficacy.

Expression of transduced carcinoembryonic antigen gene in human rhabdomyosarcoma inhibits metastasis.

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface glycoprotein belonging to the immunoglobulin superfamily. CEA has been involved in vitro in adhesion mechanisms, but little is known about the function of this glycoprotein in vivo in normal tissue differentiation and malignancy. With regard to the relationship between CEA expression and tissue differentiation, it has been reported that transfection of the CEA gene in rat L6 myoblasts results in a complete block of myogenic differentiation. To extend investigations to the transformed myogenic counterpart and examine CEA effects on differentiation and malignancy outside the colon system, we have transfected the human CEA gene in human rhabdomyosarcoma cells. Human rhabdomyosarcoma cells transfected with the CEA gene correctly expressed membrane CEA anchored via glycosylphosphatidylinositol and secreted CEA in the medium. CEA gene transfer in human rhabdomyosarcoma cells, which display a limited differentiation ability, does not further inhibit myogenic differentiation or alter in vitro proliferation or natural killer sensitivity. CEA transfection does not affect s.c. growth in nude mice, but the ectopic expression of CEA in human rhabdomyosarcoma cells can strongly inhibit their metastatic ability to lungs and adrenals after i.v. injection. The impairment of metastatic potential correlates with a reduction in the homotypic adhesion properties of the cells. These data suggest that CEA, in some systems, can interfere with intercellular adhesion and, at least for cells not metastatic to the liver, can act as an anti-metastatic molecule.

Oxygen radicals induce stress proteins and tolerance to oxidative stress in human lymphocytes.

A set of eight proteins is induced in peripheral blood lymphocytes from normal donors by exposure to hydrogen peroxide or to xanthine oxidase plus hypoxanthine. Four of them (hsp90, hsp72 and proteins 65 and 50 kDa) are also expressed after heat shock, together with proteins 110, 100 and 38 kDa. Among proteins induced after oxidative stress is a 32 kDa protein-probably corresponding to heme oxygenase-1 (HO-1)- and a 27 kDa protein, both known to be induced by reactive oxygen species. Although ionizing radiation is known to generate a number of pro-oxidant intermediates, using our one-dimensional electrophoresis system we can detect no differences in the proteins synthesized after exposure to gamma-ray doses between 5 and 20 Gy as compared with control cells. Pre-exposure to a mild hyperthermia or to moderate oxidative stress significantly increases survival of lymphocytes challenged with high doses of reactive oxygen species, in conditions compatible with a protective rôle exerted by stress proteins. The increase in survival is accompanied by the maintenance of the proliferative capacity of the cells. The physiological rôle played by stress proteins in prevention and repair of damage and the relationships between stress protein induction, oxidative state, proliferation and mode of cell death are discussed.

Antisense epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells.

Human rhabdomyosarcoma cells express membrane epidermal growth factor receptor (ECF-R), which could confer responsiveness to EGF and transforming growth factor-alpha (TGF-alpha) of autocrine or paracrine origin. To study the role played by this growth factor circuit in the proliferation and differentiation of myogenic neoplastic cells, human rhabdomyosarcoma EGF-R-expressing cells (RD/18 clone) have been transfected with a plasmid containing a fragment of the EGF-R cDNA in the antisense orientation. In vitro growth and differentiative ability were studied on six antisense-transfected clones (AS) in comparison to parental RD/18 cells and to cells transfected with the plasmid containing only the neomycin resistance gene (NEO). A reduced EGF-R membrane expression was found in AS clones by decreased immunofluorescence with an anti-EGF-R monoclonal antibody. All AS transfectants had a greatly impaired proliferative ability, even when cultured in fetal bovine serum-containing medium. Proliferation of AS clones was completely blocked in medium supplemented with 2% horse serum. The differentiation ability of AS clones was heterogeneous, ranging from clones with a percentage of myosin-positive cells higher than controls to clones with a negligible myosin expression. Therefore, the growth impairment determined by the loop interruption is not sufficient to switch on the differentiation program. The role played by EGF-R in the proliferation of human rhabdomyosarcoma cells suggests that this receptor could constitute a target for a therapeutic approach.