Photo-repair effect of a bacterial Antarctic CPD-photolyase on UVC-induced DNA lesions in human keratinocytes.

Exposure to ultraviolet radiation from sunlight induces oxidative DNA lesions and bipyrimidine photoproducts that can lead to photo-aging and skin carcinogenesis. CPD-photolyases are flavoproteins that repair cyclobutane pyrimidine dimers using blue light as an energy source. In the present work, we evaluated the photo-repair effect of the recombinant CPD-photolyase PhrAHym from the Antarctic bacterium Hymenobacter sp. UV11 on DNA lesions in human keratinocytes induced by UVC light. By performing immunochemistry assays we observed that PhrAHym repairs in a highly efficient way the CPD-photoproducts and reduces the γH2AX formation. Since this enzyme is non-cytotoxic and repairs UVC-induced DNA lesions in human keratinocytes, we propose that PhrAHym could be used as a biotherapeutic agent against UV-induced skin cancer, photoaging, and related diseases.

Neuroprotective effects of violacein in a model of inherited amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.

Lab-made 3D printed stoppers as high-throughput cell migration screening tool.

Cell migration is a process that underlies the development and maintenance of multicellular organisms, with profound implications in various pathologies. The study of cell migration is fundamental in various fields of basic biology and pharmaceutical development. Wound healing assay is an indirect way to assess cell migration. Conventional methods, such as the scratch test, are inexpensive and easy to execute but have the disadvantages of being poorly reproducible and difficult to perform on a high-throughput scale. Meanwhile, commercial strategies are expensive. In the present work, we developed a lab-made wound healing assay device that is inexpensive, easy to handle, and reproducible. We designed 3D-printed stoppers compatible with cell culture in 96-well plates. These stoppers did not affect HaCaT cells viability. The stopper-produced initial wound size was reproducible on a high-throughput scale. Also, stoppers demonstrated their effectiveness to evaluate cell migration and allowed differentiating treatments with and without fetal bovine serum. Finally, proliferation assay was determined in this wound healing model. In conclusion, our lab-made 3D-printed stopper-based assay is a more economical alternative to currently available strategies for developing reproducible, high-throughput assays to assess cell migration and proliferation.

Central Alteration in Peripheral Neuropathy of Trembler-J Mice: Hippocampal pmp22 Expression and Behavioral Profile in Anxiety Tests.

Charcot-Marie-Tooth (CMT) type 1 disease is the most common human hereditary demyelinating neuropathy. Mutations in pmp22 cause about 70% of all CMT1. Trembler-J (TrJ/+) mice are an animal model of CMT1E, having the same spontaneous pmp22 mutation that is found in humans. We compared the behavior profile of TrJ/+ and +/+ (wild-type) in open-field and elevated-plus-maze anxiety tests. In these tests, TrJ/+ showed an exclusive head shake movement, a lower frequency of rearing, but a greater frequency of grooming. In elevated-plus-maze, TrJ/+ defecate more frequently, performed fewer total entries, and have fewer entries to closed arms. These hippocampus-associated behaviors in TrJ/+ are consistent with increased anxiety levels. The expression of pmp22 and soluble PMP22 were evaluated in E17-hippocampal neurons and adult hippocampus by in situ hybridization and successive immunohistochemistry. Likewise, the expression of pmp22 was confirmed by RT-qPCR in the entire isolated hippocampi of both genotypes. Moreover, the presence of aggregated PMP22 was evidenced in unmasked granular hippocampal adult neurons and shows genotypic differences. We showed for the first time a behavior profile trait associated with anxiety and a differential expression of pmp22/PMP22 in hippocampal neurons of TrJ/+ and +/+ mice, demonstrating the involvement at the central level in an animal model of peripheral neuropathy (CMT1E).

Identification of a non-classical three-dimensional nuclear localization signal in the intestinal fatty acid binding protein.

The intestinal fatty acid binding protein (FABP) is a small protein expressed along the small intestine that bind long-chain fatty acids and other hydrophobic ligands. Several lines of evidence suggest that, once in the nucleus, it interacts with nuclear receptors, activating them and thus transferring the bound ligand into the nucleus. Previous work by our group suggests that FABP2 would participate in the cytoplasm-nucleus translocation of fatty acids. Because the consensus NLS is absent in the sequence of FABP2, we propose that a 3D signal could be responsible for its nuclear translocation. The results obtained by transfection assays of recombinant wild type and mutated forms of Danio rerio Fabp2 in Caco-2 cell cultures, showed that lysine 17, arginine 29 and lysine 30 residues, which are located in the helix-turn-helix region, would constitute a functional non-classical three-dimensional NLS.

A natural occurring bifunctional CPD/(6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9.

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme. KEY POINTS: • We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.

Calcium triggers the dissociation of myosin-Va from ribosomes in ribonucleoprotein complexes.

The sorting of RNAs to specific regions of the cell for local translation represents an important mechanism directing protein distribution and cell compartmentalization. While significant progress has been made in understanding the mechanisms underlying the transport and localization of mRNAs, the mechanisms governing ribosome mobilization are less well understood. Ribosomes present in the cytoplasm of multiple cell types can form ribonucleoprotein complexes that also contain myosin-Va (Myo5a), a processive, actin-dependent molecular motor. Here, we report that Myo5a can be disassociated from ribosomes when ribonucleoprotein complexes are exposed to calcium, both in vitro and in vivo. We suggest that Myo5a may act as a molecular switch able to anchor or release ribosomes from the actin cytoskeleton in response to intracellular signaling.

Association of microtubules and axonal RNA transferred from myelinating Schwann cells in rat sciatic nerve.

Transference of RNAs and ribosomes from Schwann cell-to-axon was demonstrated in normal and regenerating peripheral nerves. Previously, we have shown that RNAs transfer is dependent on F-actin cytoskeleton and Myosin Va. Here, we explored the contribution of microtubules to newly synthesized RNAs transport from Schwann cell nuclei up to nodal microvilli in sciatic nerves. Results using immunohistochemistry and quantitative confocal FRET analysis indicate that Schwann cell-derived RNAs co-localize with microtubules in Schwann cell cytoplasm. Additionally, transport of Schwann cell-derived RNAs is nocodazole and colchicine sensitive demonstrating its dependence on microtubule network integrity. Moreover, mRNAs codifying neuron-specific proteins are among Schwann cell newly synthesized RNAs population, and some of them are associated with KIF1B and KIF5B microtubules-based motors.

Neuroprotective effects of novel nitrones: In vitro and in silico studies.

Neurodegenerative diseases affect millions of people around the world. Several studies point out caspase-3 as a key player in the development and progression of neurological disorders including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's and Huntington's diseases. Furthermore, oxidative stress and mitochondrial dysfunction plays an important role in neurodegenerative pathologies leading to neuronal damage and cell death. Pharmacological properties of nitrones such as free radical trapping and neuroprotection has been previously described. In the present work, we have assessed ten non-cytotoxic nitrones for their ability to inhibit apoptosis plus their potential to reduce active caspase-3 and oxidative stress in the hippocampal neuronal cell line HT22. Our results highlight the faculty of nitrones to inhibit apoptosis by a mechanism that involves active caspase-3 reduction and decrease of reactive oxygen species. Moreover, docking and molecular dynamics approaches lead to a detailed analysis at the atomic level of the nitrones binding mode to caspase-3 suggesting that compounds bind in a region close to the catalytic site. All these data place these molecules as excellent hits for further efforts to redesign novel compounds in the search of a new therapy against neurodegenerative disorders.

Furoxans and tocopherol analogs-furoxan hybrids as anticancer agents.

We determined the antiproliferative and nitric oxide (NO)-releasing activity of furoxans and tocopherol analogs-furoxan hybrids by tandem Griess/resazurin/sulforhodamin B assays in HeLa, 253J, T24, and HepG2 cancer cells. In addition, to investigate the NO implications in the inhibition of cell growth, cells were pretreated with the NO scavenger hemoglobin and the genotoxic damage was determined. The compounds 1 and 3 emerged as good anticancer agents for bladder cancer treatment. The NO-releasing activity of these compounds appears to be necessary to obtain the antiproliferative effect. Although compound 1 exerted a DNA damage mechanism of action, compound 3 seemed to act in a different way. The low toxicity levels against normal cell line HaCaT point them out as a very promising scaffold for the further design of new anticancer agents.

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei.

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC(12) but not BODIPY-FLC(5) to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC(12) to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC(12) was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity.

Myosin Va associates with mRNA in ribonucleoprotein particles present in myelinated peripheral axons and in the central nervous system.

Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems.

Association of Myosin Va and Schwann cells-derived RNA in mammal myelinated axons, analyzed by immunocytochemistry and confocal FRET microscopy.

Evidence from multiple sources supports the hypothesis that Schwann cells in the peripheral nervous system transfer messenger RNA and ribosomes to the axons they ensheath. Several technical and methodological difficulties exist for investigators to unravel this process in myelinated axons - a complex two-cell unit. We present an experimental design to demonstrate that newly synthesized RNA is transferred from Schwann cells to axons in association with Myosin Va. The use of quantitative confocal FRET microscopy to track newly-synthesized RNA and determine the molecular association with Myosin Va, is described in detail.

Glia to axon RNA transfer.

The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial-to-axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024-11029; Court et al. [2011]: Glia 59:1529-1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased.

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

F-actin distribution at nodes of Ranvier and Schmidt-Lanterman incisures in mammalian sciatic nerves.

Very little is known about the function of the F-actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F-actin at Schmidt-Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F-actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F-actin, as compared with normal nerves. Cytochalasin-D and Latrunculin-A were used to disrupt the F-actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F-actin, but in different ways. Cytochalasin-D did not disrupt Schwann cell (SC) F-actin at the NR. Latrunculin-A did not disrupt F-actin at the boundary region between SC and axon at the NR domain. We surmise that the rearrangement of F-actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a Charcot-Marie-Tooth (CMT) model.

Localization of mRNA in vertebrate axonal compartments by in situ hybridization.

The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.

Early phenotypical diagnoses in Trembler-J mice model.

Pmp-22 mutant mice (Trembler-J: B6.D2-Pmp22/J), are used as a model to study Charcot-Marie-Tooth type 1A (CMT1A). The identification of individual genotypes is a routine in the management of the Tr(J) colony. The earliest phenotypic manifestation of the pmp-22 mutation is just about 20th postnatal days, when pups begin to tremble. In this study, a rapid and simple diagnostic method was developed by modifying the Tail Suspension Test (MTST) to determine the difference between the Tr(J) and the wild-type mice phenotype. The animal behavioral phenotypes generated during the test were consistent with the specific genotype of each animal. The MTST allowed us to infer the heterozygous genotype in early postnatal stages, at 11 days after birth. The motor impairment of Tr(J) mice was also analyzed by a Fixed Bar Test (FBT), which revealed the disease evolution according to age. The main advantages of MTST are its objectivity, simplicity, and from the viewpoint of animal welfare, it is a non-invasive technique that combined with his rapidity show its very well applicability for use from an early age in these mice.

Two novel Mesocestoides vogae fatty acid binding proteins--functional and evolutionary implications.

This work describes two new fatty acid binding proteins (FABPs) identified in the parasite platyhelminth Mesocestoides vogae (syn. corti). The corresponding polypeptide chains share 62% identical residues and overall 90% similarity according to CLUSTALX default conditions. Compared with Cestoda FABPs, these proteins share the highest similarity score with the Taenia solium protein. M. vogae FABPs are also phylogenetically related to the FABP3/FABP4 mammalian FABP subfamilies. The native proteins were purified by chromatographical procedures, and apparent molecular mass and isoelectric point were determined. Immunolocalization studies determined the localization of the expression of these proteins in the larval form of the parasite. The genomic exon-intron organization of both genes is also reported, and supports new insights on intron evolution. Consensus motifs involved in splicing were identified.

Preliminary analysis of cold stress responsive proteins in Mesocestoides corti larvae.

Many parasites undergo sudden changes in environmental conditions at some stage during their life cycle. The molecular response to this variation is characterised by a rapid transcriptional activation of a specific set of genes coding for proteins generically known as stress proteins. They appear to be also involved in various biological processes including cell proliferation and differentiation. The platyhelminth parasite, Mesocestoides corti (Cestoda) presents important properties as a model organism. Under stress conditions, key molecules involved in metabolic pathways as well as in the growth and differentiation of the parasite can be identified. 2D protein expression profile of tetrathyridia of M. corti, submitted to nutritional starvation and cold stress is described, as well as the recovery pattern. A set of specifically expressed proteins was observed in each experimental condition. Quantitative and qualitative differences and stress recovery pattern are also reported. This work makes evident the high plasticity and resistance to extreme environmental conditions of these parasites at the molecular level.