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Submitted by Paula Luz (pluz@ualg.pt) on 2021-11-27T11:38:46Z No. of bitstreams: 1 Draft Genome Sequence of Vibrio jasicida 20LP, an.pdf: 256421 bytes, checksum: 193013f45c8d4aefc3d07c07f9c3358c (MD5) Approved for entry into archive by Sofia Lopes (silopes@ualg.pt) on 2021-12-02T19:30:23Z (GMT) No. of bitstreams: 1 Draft Genome Sequence of Vibrio jasicida 20LP, an.pdf: 256421 bytes, checksum: 193013f45c8d4aefc3d07c07f9c3358c (MD5) Made available in DSpace on 2021-12-02T19:30:24Z (GMT). No. of bitstreams: 1 Draft Genome Sequence of Vibrio jasicida 20LP, an.pdf: 256421 bytes, checksum: 193013f45c8d4aefc3d07c07f9c3358c (MD5) Previous issue date: 2021-11 info:eu-repo/semantics/publishedVersion

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Here, we announce the genomes of eight Roseibium album (synonym Labrenzia alba ) strains that were obtained from the octocoral Eunicella labiata . Genome annotation revealed multiple symbiosis factors common to all genomes, such as eukaryotic-like repeat protein- and multidrug resistance-encoding genes, which likely underpin symbiotic relationships with marine invertebrate hosts.

Deafferentation is an important determinant of plastic changes in the CNS, which consists of a loss of inputs from the body periphery or from the CNS itself. Although cortical reorganization has been well documented, white matter plasticity was less explored. Our goal was to investigate microstructural interhemispheric connectivity changes in early and late amputated rats. For that purpose, we employed diffusion-weighted magnetic resonance imaging, as well as Western blotting, immunohistochemistry, and electron microscopy of sections of the white matter tracts to analyze the microstructural changes in the corticospinal tract and in the corpus callosum (CC) sector that contains somatosensory fibers integrating cortical areas representing the forelimbs and compare differences in rats undergoing forelimb amputation as neonates, with those amputated as adults. Results showed that early amputation induced decreased fractional anisotropy values and reduction of total myelin amount in the cerebral peduncle contralateral to the amputation. Both early and late forelimb amputations induced decreased myelination of callosal fibers. While early amputation affected myelination of thinner axons, late amputation disrupted axons of all calibers. Since the CC provides a modulation of inhibition and excitation between the hemispheres, we suggest that the demyelination observed among callosal fibers may misbalance this modulation.

Peripheral blood sample was donated by a 61 years old female patient diagnosed with acute myeloid leukemia secondary to a primary myelofibrosis harboring the 52-bp deletion in the CALR gene (c.1092_1143del, p.L367fs*46) and the R693X mutation in the ASXL1 gene (c.2077C>T, p.R693X). CD34+ cells were isolated from the sample and subjected to the reprogramming procedure by using the Sendai virus carrying the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc. iPS colonies generated retained the original mutations and displayed all the features of bona fide iPS cells.

Zika virus (ZIKV) has been extensively studied since it was linked to congenital malformations, and recent research has revealed that astrocytes are targets of ZIKV. However, the consequences of ZIKV infection, especially to this cell type, remain largely unknown, particularly considering integrative studies aiming to understand the crosstalk among key cellular mechanisms and fates involved in the neurotoxicity of the virus. Here, the consequences of ZIKV infection in iPSC-derived astrocytes are presented. Our results show ROS imbalance, mitochondrial defects and DNA breakage, which have been previously linked to neurological disorders. We have also detected glial reactivity, also present in mice and in post-mortem brains from infected neonates from the Northeast of Brazil. Given the role of glia in the developing brain, these findings may help to explain the observed effects in congenital Zika syndrome related to neuronal loss and motor deficit. ispartof: SCIENTIFIC REPORTS vol:10 issue:1 ispartof: location:England status: published

Primary myelofibrosis (PMF) is a hematological malignancy characterized by activation of the JAK/STAT pathway and risk of leukemic transformation. In this study, we generated an induced Pluripotent Stem (iPS) cell line derived from a 65-year old male PMF patient carrying the 5-pb insertion in the CALR gene (CALRins5) and the c.437 G > A mutation in the TP53 gene (p.W146X). The newly derived PMF3.17 iPS cell line harbors the original mutations and was characterized as bona fide iPS. Resource table Unique stem cell line identifier INCABRi002-A Alternative name(s) of stem cell line PMF3.17 Institution Brazilian National Cancer Institute and D'Or Institute for Research and Education Contact information of distributor Martin Bonamino, PhD, mbonamino@inca.gov.br , Barbara Monte Mor, PhD, barbara.montemor@inca.gov.br , Stevens Rehen, PhD, srehen@lance-ufrj.org Type of cell line iPS cell Origin Human Additional origin info Age: 65 Sex: male Cell source Peripheral blood Clonality Clonal Method of reprogramming Sendai Virus Genetic modification No Type of modification Not applicable Associated disease Primary myelofibrosis Gene CALR gene: CALRins5, c.1154_1155insTTGTC, p.K385 fs*47 TP53 gene: c.437 G > A, p.W146X Method of modification Not applicable Name of transgene or resistance Not applicable Inducible/constitutive system Not applicable Date archived/stock date July 7th, 2015 Cell line repository/bank Not applicable Ethical approval Ethics Committee of the Brazilian National Cancer Institute (INCA) under the number 062/08. Ethics Review Board-competent authority obtained.

Oligodendrocytes (OLs) are responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). This protocol describes a method to generate oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells (hPSCs) in only ~20 d, which can subsequently myelinate neurons, both in vitro and in vivo. To date, OPCs have been derived from eight different hPSC lines including those derived from patients with spontaneous and familial forms of MS and ALS, respectively. hPSCs, fated for 8 d toward neural progenitors, are transduced with an inducible lentiviral vector encoding for SOX10. The addition of doxycycline for 10 d results in60% of cells being O4-expressing OPCs, of which 20% co-express the mature OL marker myelin basic protein (MBP). The protocol also describes an alternative for viral transduction, by incorporating an inducible SOX10 in the safe harbor locus AAVS1, yielding ~100% pure OPCs. O4

Neural development represents a dynamic process where mitochondrial integrity is decisive for neuronal activity. Structural changes in these organelles may be related to neurological disorders. Valproic acid (VPA) is an anticonvulsive drug commonly used for epilepsy treatment and its use is associated to increased risk of neuropsychiatric disorders. Recently we showed changes in shape and membrane potential in mitochondria from human neural progenitor cells (NPCs) exposed to VPA (da Costa et al. 2015). Here, we applied transmission electron microscopy and electron tomography to evaluate mitochondrial damage caused by VPA in NPCs. Results showed mitochondrial cristae disorganization in a dose dependent manner. Disturbance in mitochondrial ultrastructure may influence metabolism, leading to synaptic plasticity and neurogenesis impairment. These data contribute to understanding VPA exposure potential effects on brain development.

Systematic studies of micronutrients during brain formation are hindered by restrictions to animal models and adult post-mortem tissues. Recently, advances in stem cell biology have enabled recapitulation of the early stages of human telencephalon developmentin vitro. In the present work, we analyzed cerebral organoids derived from human pluripotent stem cells by synchrotron radiation X-ray fluorescence in order to measure biologically valuable micronutrients incorporated and distributed into the exogenously developing brain. Our findings indicate that elemental inclusion in organoids is consistent with human brain tissue and involves P, S, K, Ca, Fe and Zn. Occurrence of different concentration gradients also suggests active regulation of elemental transmembrane transport. Finally, the analysis of pairs of elements shows interesting elemental interaction patterns that change from 30 to 45 days of development, suggesting short- or long-term associations, such as storage in similar compartments or relevance for time-dependent biological processes. These findings shed light on which trace elements are important during human brain development and will support studies aimed to unravel the consequences of disrupted metal homeostasis for neurodevelopmental diseases, including those manifested in adulthood.

2,4-Dinitrophenol (DNP) is a neuroprotective compound previously shown to promote neuronal differentiation in a neuroblastoma cell line and neurite outgrowth in primary neurons. Here, we tested the hypothesis that DNP could induce neurogenesis in embryonic stem cells (ESCs). Murine ESCs, grown as embryoid bodies (EBs), were exposed to 20μM DNP (or vehicle) for 4days. Significant increases in the proportion of nestin- and β-tubulin III-positive cells were detected after EB exposure to DNP, accompanied by enhanced glial fibrillary acidic protein (GFAP), phosphorylated extracellular signal-regulated kinase (p-ERK) and ATP-linked oxygen consumption, thought to mediate DNP-induced neural differentiation. DNP further protected ESCs from cell death, as indicated by reduced caspase-3 positive cells, and increased proliferation. Cell migration from EBs was significantly higher in DNP-treated EBs, and migrating cells were positive for nestin, ß-tubulin III and MAP2, similar to that observed with retinoic acid (RA)-treated EBs. Compared to RA, however, DNP exerted a marked neuritogenic effect on differentiating ESCs, increasing the average length and number of neurites per cell. Results establish that DNP induces neural differentiation of ESCs, accompanied by cell proliferation, migration and neuritogenesis, suggesting that DNP may be a novel tool to induce neurogenesis in embryonic stem cells.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil / University of Rio de Janeiro. Institute of Biomedical Sciences. Rio de Janeiro, RJ, Brazil. D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil / University of Campinas. Institute of Biology. Department of Biochemistry and Tissue Biology. Campinas, SP, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil. Federal University of Rio de Janeiro. Institute of Biology. Rio de Janeiro, RJ, Brazil. D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil. D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil. Federal University of Rio de Janeiro. Institute of Biology. Rio de Janeiro, RJ, Brazil. University of Campinas. Institute of Biology. Department of Biochemistry and Tissue Biology. Campinas, SP, Brazil. D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Institute Oswaldo Cruz. Rio de Janeiro, RJ, Brazil. D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil. Federal University of Rio de Janeiro. Institute of Biology. Rio de Janeiro, RJ, Brazil. Federal University of Par?. Bel?m, PA, Brazil. Funda??o Oswaldo Cruz. Institute Oswaldo Cruz. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Federal University of Rio de Janeiro. Institute of Biology. Rio de Janeiro, RJ, Brazil. University of Campinas. Institute of Biology. Department of Biochemistry and Tissue Biology. Campinas, SP, Brazil. D?Or Institute for Research and Education. Rio de Janeiro, RJ, Brazil / University of Rio de Janeiro. Institute of Biomedical Sciences. Rio de Janeiro, RJ, Brazil. Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular and cellular consequences of Zika virus circulating in Brazil to the human brain development have not been studied yet. Here we describe alterations in human neurospheres derived from neural stem cells infected with Brazilian ZIKV. Combined proteomics and mRNA transcriptional profile analyses showed that Brazilian ZIKV, prior to induce cell death, alters cell cycle and halts neurogenic programmes, in addition to regulate transcription and protein translation due to viral replication. These results point to biological mechanisms potentially implicated in brain malformations as a result of ZIKV congenital infection.

Zika virus tested in human brain organoids The pernicious and resilient Aedes mosquito is rapidly spreading Zika virus (ZIKV) through the Americas. ZIKV infection mostly causes mild disease, but in some patients, nervous system involvement is indicated. A particular worry is an observed correlation between infection of mothers in the first trimester of pregnancy and microcephaly in newborns. Garcez et al. tested the effects of ZIKV compared with dengue virus infection on human neural stem cells grown as organoids. ZIKV targeted the human brain cells, reduced their size and viability in vitro, and caused programmed cell death responses. Science , this issue p. 816

Immature neurons migrate tangentially within the rostral migratory stream (RMS) to the adult olfactory bulb (OB), then radially to their final positions as granule and periglomerular neurons; the controls over this transition are not well understood. Using adult transgenic mice with the human GFAP promoter driving expression of enhanced GFP, we identified a population of radial glia-like cells that we term adult olfactory radial glia-like cells (AORGs). AORGs have large, round somas and simple, radially oriented processes. Confocal reconstructions indicate that AORGs variably express typical radial glial markers, only rarely express mouse GFAP, and do not express astroglial, oligodendroglial, neuronal, or tanycyte markers. Electron microscopy provides further supporting evidence that AORGs are not immature neurons. Developmental analyses indicate that AORGs are present as early as P1, and are generated through adulthood. Tracing studies show that AORGs are not born in the SVZa, suggesting that they are born either in the RMS or the OB. Migrating immature neurons from the adult SVZa are closely apposed to AORGs during radial migration in vivo and in vitro. Taken together, these data indicate a newly-identified population of radial glia-like cells in the adult OB that might function uniquely in neuronal radial migration during adult OB neurogenesis.

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca2+ concentration from 0.05 up to 0.70 μm. It is not known how the rise of Ca2+ concentration activates BAT thermogenesis. In this report we compared the effects of Ca2+ in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca2+-ATPase bound to the inner membrane of BAT mitochondria. A Ca2+-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca2+ enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca2+ concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 μm. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca2+-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca2+ did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca2+-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca2+ concentrations similar to those noted in the cell during adrenergic stimulation.

The effects of the microtubule affecting drugs taxol, nocodazole and colchicine on the cell cycle and ultrastructure of Tritrichomonas foetus, a protist parasite of cattle, were studied. Alterations in the cytoskeleton, motility and organellar ultrastructure were followed using anti-tubulin antibodies and fluorescence microscopy, scanning- and transmission-electron microscopy. Flow cytometry was also used to analyze the effect of the drugs on the cell cycle. T. foetus was treated with 10 microM taxol, 15 microM nocodazole or 1.5 mM colchicine for 12 h. The first effect observed was pseudocyst formation and alterations in cell motility. The cell cycle was affected and the cells have blocked cytokinesis, but not karyokinesis. The behavior of Golgi, hydrogenosomes and vacuoles was analyzed. The following effects were seen following drug treatments: (1) cell motility was altered and flagella internalized; (2) microtubules of the pelta-axostyle complex were not depolymerized and the axostyle assumed a curved form; (3) hydrogenosomes were of abnormal size and shape; (4) cells became multinucleate; (5) the division process was blocked in cytokinesis; (6) autophagic vacuoles containing a large amount of microtubules were seen; (7) axoneme organization was altered; (8) zoids were formed; (9) signs of cell death, such as membrane blebbing, were observed.

The axon is a highly specialized compartment of neurons. Besides their basic function connecting neurons to their targets, axons play key roles in the nervous system. They are involved in the transport of several molecules indispensable to neuronal activity, act as sensors to guidance cues during development and regeneration, and are essential to maintain normal glial cell functions and myelin sheath assembly (Nave & Trap 2008). Recent evidence indicates that mRNA and Schwann cells-delivered ribosomes can be found within the axoplasm, suggesting that axons may be capable of synthesizing specific proteins (Court et al., 2008). However, most axonal structural proteins are synthesized in the neuronal cell body and transported along the length of the axon. Interruption of this supply leads to a degenerative process known as Wallerian degeneration (WD) in the distal portion of the axon (Coleman, 2005). WD is triggered by intrinsic degenerative pathways that are not correlated to cellular apoptosis (Finn et al., 2000). Axon degeneration is a final common pathway observed not only after a traumatic nerve injury, but also in many neurodegenerative disorders (e.g., Parkinson`s and Alzheimer`s diseases) and in demyelinating diseases such as multiple sclerosis (Coleman, 2005; Coleman & Freeman, 2010). Uncovering the mechanisms that trigger and control axon degeneration is extremely relevant, as such knowledge may offer novel tools to treat severed or damaged axons as well as several neurodegenerative disorders in which WD takes place. In this chapter, we will review the basic concepts of WD, with emphasis on the mechanisms that control axon degeneration following trauma. Next, we will address the issue whether or not current antidegenerative strategies are efficient and can be envisioned to be applied to humans in the near future.

BackgroundThe helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space.Principal findingsWe showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated.ConclusionsWe showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

BackgroundTumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls.Methodology/principal findingsVarious cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed that NaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O(2) consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged.ConclusionNaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype.

Preventing the harm caused by nerve degeneration is a major challenge in neurodegenerative diseases and in various forms of trauma to the nervous system. The aim of the current work was to investigate the effects of systemic administration of 2,4-dinitrophenol (DNP), a compound with newly recognized neuroprotective properties, on sciatic-nerve degeneration following a crush injury. Sciatic-nerve injury was induced by unilateral application of an aneurysm clip. Four groups of mice were used: uninjured, injured treated with vehicle (PBS), injured treated with two intraperitoneal doses of DNP (0.06 mg DNP/kg every 24 h), and injured treated with four doses of DNP (every 12 h). Animals were sacrificed 48 h post injury and both injured and uninjured (contralateral) sciatic nerves were processed for light and electron microscopy. Morphometric, ultrastructural, and immunohistochemical analysis of injured nerves established that DNP prevented axonal degeneration, blocked cytoskeletal disintegration, and preserved the immunoreactivity of amyloid precursor protein (APP) and Neuregulin 1 (Nrg1), proteins implicated in neuronal survival and myelination. Functional tests revealed preservation of limb function following injury in DNP-treated animals. Results indicate that DNP prevents nerve degeneration and suggest that it may be a useful small-molecule adjuvant in the development of novel therapeutic approaches in nerve injury.

Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca(2+)-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+) effect in BAT mitochondria thermogenesis. We found that Ca(2+) increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+) concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+) strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+) activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.

The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation.

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca(2+) concentration from 0.05 up to 0.70 mum. It is not known how the rise of Ca(2+) concentration activates BAT thermogenesis. In this report we compared the effects of Ca(2+) in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase bound to the inner membrane of BAT mitochondria. A Ca(2+)-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca(2+) enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca(2+) concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 mum. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca(2+) did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca(2+)-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca(2+) concentrations similar to those noted in the cell during adrenergic stimulation.

Trichomonas vaginalis, a protist parasite of the urogenital tract in humans, is the causative agent of trichomonosis, which in recent years have been associated with the cervical cancer development. In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T. vaginalis and the influence of the iron concentration present in the parasite's culture medium on the interaction effects. Our results show that T. vaginalis adheres to the epithelial cell causing alterations in the junctional complex, such as: (a) a decrease in transepithelial electrical resistance; (b) alteration in the pattern of junctional complex proteins distribution as observed for E-cadherin, occludin and ZO-1; and (c) enlargement of the spaces between epithelial cells. These effects were dependent on (a) the degree of the parasite virulence isolate, (b) the iron concentration in the culture medium, and (c) the expression of adhesin proteins on the parasite surface.

The Ca2+-ATPase (SERCA) found in vesicles derived from the sarco/endoplasmic reticulum vesicles of rats brown adipose tissue and rabbit white muscle were identified by gel electrophoresis, Western blot, electron microscopy and immunolabeling with gold particles. In both tissues, the isoform found was SERCA 1. The Ca2+ affinity of the fat SERCA 1 was different from the muscle isoform. The degree of uncoupling is estimated measuring the ratio between Ca2+ transport and ATP cleaved. In brown fat vesicles the degree of uncoupling varied depending on the Ca2+ concentration of the medium. This was not observed in vesicles derived from muscle. At all Ca2+ concentrations tested, the uncoupling was not related to Ca2+ leakage from the membrane and was far more pronounced in fat than in muscle vesicle. When a Ca2+ gradient was formed across the vesicles membrane the heat released during ATP hydrolysis varied between 22 and 26 Kcal/mol in both fat and muscle vesicles but in the absence of a gradient the heat released was 17 Kcal/mol in fat and 12 Kcal/mol in muscle. The data reported indicate that the SERCA 1 of brown adipocytes is far more thermogenic than the white muscle SERCA 1, and suggest that, in addition to storing Ca2+ inside the endoplasmic reticulum, the SERCA 1 may represent a source of heat production contributing to the thermogenic function of brown adipose tissue.