ArtinM Cytotoxicity in B Cells Derived from Non-Hodgkin's Lymphoma Depends on Syk and Src Family Kinases.

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin's lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin's lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.

Melanoma spheroid-containing artificial dermis as an alternative approach to in vivo models.

Melanoma spheroid-loaded 3D skin models allow for the study of crucial tumor characteristics and factors at a superior level because the neoplastic cells are integrated into essential human skin components, permitting tumor-skin model communication. Herein, we designed a melanoma-containing artificial dermis by inserting multicellular tumor spheroids from the metastatic phase of WM 1617 melanoma cells into an artificial dermis. We cultured multicellular melanoma spheroids by hanging drop method (250 cells per drop) with a size of 420 µm in diameter after incubation for 14 days. These spheroids were integrated into the dermal equivalents that had been previously preparedwith a type-I collagen matrix and healthy fibroblasts. The melanoma spheroid cells invaded and proliferated in the artificial dermis. Spheroids treated with a 1.0 µmol/L aluminum chloride phthalocyanine nanoemulsion in the absence of light showed high cell viability. In contrast, under irradiation with visible red light (660 nm) at 25 J/cm2, melanoma cells were killed and the healthy tissue was preserved, indicating that photodynamic therapy is effective in such a model. Therefore, the 3D skin melanoma model has potential to promote research in full-thickness skin model targeting optimized preclinical assays.

A Genetic Model of Epilepsy with a Partial Alzheimer's Disease-Like Phenotype and Central Insulin Resistance.

Studies have suggested an important connection between epilepsy and Alzheimer's disease (AD), mostly due to the high number of patients diagnosed with AD who develop epileptic seizures later on. However, this link is not well understood. Previous studies from our group have identified memory impairment and metabolic abnormalities in the Wistar audiogenic rat (WAR) strain, a genetic model of epilepsy. Our goal was to investigate AD behavioral and molecular alterations, including brain insulin resistance, in naïve (seizure-free) animals of the WAR strain. We used the Morris water maze (MWM) test to evaluate spatial learning and memory performance and hippocampal tissue to verify possible molecular and immunohistochemical alterations. WARs presented worse performance in the MWM test (p < 0.0001), higher levels of hyperphosphorylated tau (S396) (p < 0.0001) and phosphorylated glycogen synthase kinase 3 (S21/9) (p < 0.05), and lower insulin receptor levels (p < 0.05). Conversely, WARs and Wistar controls present progressive increase in amyloid fibrils (p < 0.0001) and low levels of soluble amyloid-ß. Interestingly, the detected alterations were age-dependent, reaching larger differences in aged than in young adult animals. In summary, the present study provides evidence of a partial AD-like phenotype, including altered regulation of insulin signaling, in a genetic model of epilepsy. Together, these data contribute to the understanding of the connection between epilepsy and AD as comorbidities. Moreover, since both tau hyperphosphorylation and altered insulin signaling have already been reported in epilepsy and AD, these two events should be considered as important components in the interconnection between epilepsy and AD pathogenesis and, therefore, potential therapeutic targets in this field.

The IRM cell adhesion molecules Hibris, Kin of irre and Roughest control egg morphology by modulating ovarian muscle contraction in Drosophila.

The Irre Cell Recognition Module (IRM) is an evolutionarily conserved group of transmembrane glycoproteins required for cell-cell recognition and adhesion in metazoan development. In Drosophila melanogaster ovaries, four members of this group - Roughest (Rst), Kin of irre (Kirre), Hibris (Hbs) and Sticks and stones (Sns) - play important roles in germ cell encapsulation and muscle sheath organization during early pupal stages, as well as in the progression to late oogenesis in the adult. Females carrying some of the mutant rst alleles are viable but sterile, and previous work from our laboratory had identified defects in the organization of the peritoneal and epithelial muscle sheaths of these mutants that could underlie their sterile phenotype. In this study, besides further characterizing the sterility phenotype associated with rst mutants, we investigated the role of the IRM molecules Rst, Kirre and Hbs in maintaining the functionality of the ovarian muscle sheaths. We found that knocking down any of the three genes in these structures, either individually or in double heterozygous combinations, not only decreases contraction frequency but also irregularly increases contraction amplitude. Furthermore, these alterations can significantly impact the morphology of eggs laid by IRM-depleted females demonstrating a hitherto unknown role of IRM molecules in egg morphogenesis.

Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease.

BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.

Whole transcriptome analysis reveals correlation of long noncoding RNA ZEB1-AS1 with invasive profile in melanoma.

Melanoma is the deadliest form of skin cancer, and little is known about the impact of deregulated expression of long noncoding RNAs (lncRNAs) in the progression of this cancer. In this study, we explored RNA-Seq data to search for lncRNAs associated with melanoma progression. We found distinct lncRNA gene expression patterns across melanocytes, primary and metastatic melanoma cells. Also, we observed upregulation of the lncRNA ZEB1-AS1 (ZEB1 antisense RNA 1) in melanoma cell lines. Data analysis from The Cancer Genome Atlas (TCGA) confirmed higher ZEB1-AS1 expression in metastatic melanoma and its association with hotspot mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) gene and RAS family genes. In addition, a positive correlation between ZEB1-AS1 and ZEB1 (zinc finger E-box binding homeobox 1) gene expression was verified in primary and metastatic melanomas. Using gene expression signatures indicative of invasive or proliferative phenotypes, we found an association between ZEB1-AS1 upregulation and a transcriptional profile for invasiveness. Enrichment analysis of correlated genes demonstrated cancer genes and pathways associated with ZEB1-AS1. We suggest that the lncRNA ZEB1-AS1 could function by activating ZEB1 gene expression, thereby influencing invasiveness and phenotype switching in melanoma, an epithelial-to-mesenchymal transition (EMT)-like process, which the ZEB1 gene has an essential role.

The highly efficient powerhouse in the Wistar audiogenic rat, an epileptic rat strain.

The Wistar audiogenic rat (WAR) is an animal model of tonic-clonic epileptic seizures, developed after genetic selection by sister × brother inbreeding of Wistar rats susceptible to sound stimuli. Although metabolic changes have been described in this strain, nothing is known about its mitochondrial metabolism. Here, we addressed mitochondrial aspects of oxidative phosphorylation, oxidative stress, biogenesis, and dynamics in liver, skeletal muscle, and heart of male WARs and correlating them with physiological aspects of body metabolism. The results showed higher mitochondrial content, respiration rates in phosphorylation and noncoupled states, and H2O2 production in WARs. Liver presented higher content of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and mammalian target of rapamycin, proteins related to mitochondrial biogenesis. In agreement, isolated liver mitochondria from WARs showed higher respiration rates in phosphorylation state and ADP-to-O ratio, as well as higher content of proteins related to electron transport chain ATP synthase, TCA cycle, and mitochondrial fusion and fission compared with their Wistar counterparts. Mitochondria with higher area and perimeter and more variable shapes were found in liver and soleus from WARs in addition to lower reduced-to-oxidized glutathione ratio. In vivo, WARs demonstrated lower body mass and energy expenditure but higher food and water intake and amino acid oxidation. When exposed to a running test, WARs reached higher speed and resisted for a longer time and distance than their Wistar controls. In conclusion, the WAR strain has mitochondrial changes in liver, skeletal muscle, and heart that improve its mitochondrial capacity of ATP production, making it an excellent rat model to study PGC1α overexpression and mitochondrial function in different physiological conditions or facing pathological challenges.

Cytotoxicity of Structurally Diverse Anthranoids and Correlation with Mechanism of Action and Side Effects.

The purpose of this contribution is to evaluate the cytotoxicity and apoptosis inducing ability of structurally diverse anthraquinones to establish a relationship between structure and toxicity. Besides the wide spread use of anthraquinones in pharmacological drugs for constipation and non-prescription dietary supplements for weight loss, extracts are still commercialized as crude extracts and long-term side effects are still relevant. In this work we developed a method to quantify the cascarosides isolated from Rhamnus purshiana (Cascara Sagrada) using LC-MS/MS and evaluated the effects of this extract and isolated compounds on cellular viability using NOK-SI, HeLa, and T98G cell lineages. Apoptosis inducing ability was also analyzed via evaluating key-proteins involved in apoptosis pathways. Using cascarosides isolated from bark extracts, we found that the presence of glucose moieties in the chemical structure reduced the toxicity. This communication reviewed the mechanisms of action, toxicity of anthraquinones and correlated the toxicity with chemical structures of cascarosides. Results indicate that cascarosides-enriched cascara extract, as well as glycosylated anthraquinones, may have some beneficial effects for laxative action of herbal medicines. Considering our results, a cascarosides-enrichment in cascara extract is recommended.

Protective action of Omega-3 on paraquat intoxication in Drosophila melanogaster.

Paraquat (PQ) (1,1'-dimethyl-4-4'-bipyridinium dichloride) is the second most widely used herbicide worldwide; however, in countries different sales and distribution remain restricted. Chronic exposure to PQ leads to several diseases related to oxidative stress and mitochondrial dysfunctions including myocardial failure, cancer, and neurodegeneration and subsequently death depending upon the dose level. The aim of this study was to examine if diet supplementation with eicosapentaenoic and docosahexaenoic acids (EPA and DHA, omega-3 long-chain fatty acids) serves a protective mechanism against neuromuscular dysfunctions mediated by PQ using Drosophila melanogaster as a model with focus on mitochondrial metabolism. PQ ingestion (170 mg/kg b.w. for 3 d) resulted in a decreased life span and climbing ability in D. melanogaster. In the brain, PQ increased thioflavin fluorescence and reduced either 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclei staining and neuronal nuclei protein (NeuN) positive neurons, indicating amyloid formation and neurodegenetation, respectively. In the thorax, PQ ingestion lowered citrate synthase activity and respiratory functions indicating a reduction in mitochondrial content. PQ elevated Ca2+/calmodulin-dependent protein kinase II (CaMKII) mRNA expression levels, indicative of high calcium influx from cytosol to mitochondrial matrix. In brain and thorax, PQ also increased hydrogen peroxide (H2O2) production and impaired acetylcholinesterase (AChE) activity. Concomitant EPA/DHA ingestion (0.31/0.19 mg/kg b.w.) protected D. melanogaster against PQ-induced toxicity preserving neuromuscular function and slowing down the rate of aging. In brain and thorax, these omega-3 fatty acids inhibited excess H2O2 production and restored AChE activity. EPA/DHA delayed amyloid deposition in the brain, and restored low citrate synthase activity and respiratory functions in the thorax. The effects in the thorax were attributed to stimulated mRNA expression level of genes involved either in mitochondrial dynamics or biogenesis promoted by EPA/DHA: dynamin-related protein (DRP1), mitochondrial assembly regulatory factor (MARF), mitochondrial dynamin like GTPase (OPA1), and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). In conclusion, diet supplementation with EPA/DHA appears to protect D. melanogaster muscular and neuronal tissues against PQ intoxication.

Identification of Long Noncoding RNAs Deregulated in Papillary Thyroid Cancer and Correlated with BRAFV600E Mutation by Bioinformatics Integrative Analysis.

Papillary Thyroid Cancer (PTC) is an endocrine malignancy in which BRAFV600E oncogenic mutation induces the most aggressive phenotype. In this way, considering that lncRNAs are arising as key players in oncogenesis, it is of high interest the identification of BRAFV600E-associated long noncoding RNAs, which can provide possible candidates for secondary mechanisms of BRAF-induced malignancy in PTC. In this study, we identified differentially expressed lncRNAs correlated with BRAFV600E in PTC and, also, extended the cohort of paired normal and PTC samples to more accurately identify differentially expressed lncRNAs between these conditions. Indirectly validated targets of the differentially expressed lncRNAs in PTC compared to matched normal samples demonstrated an involvement in surface receptors responsible for signal transduction and cell adhesion, as well as, regulation of cell death, proliferation and apoptosis. Targets of BRAFV600E-correlated lncRNAs are mainly involved in calcium signaling pathway, ECM-receptor interaction and MAPK pathway. In summary, our study provides candidate lncRNAs that can be either used for future studies related to diagnosis/prognosis or as targets for PTC management.

Modulation of HJURP (Holliday Junction-Recognizing Protein) levels is correlated with glioblastoma cells survival.

BACKGROUND: Diffuse astrocytomas are the most common type of primary brain cancer in adults. They present a wide variation in differentiation and aggressiveness, being classified into three grades: low-grade diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV), the most frequent and the major lethal type. Recent studies have highlighted the molecular heterogeneity of astrocytomas and demonstrated that large-scale analysis of gene expression could help in their classification and treatment. In this context, we previously demonstrated that HJURP, a novel protein involved in the repair of DNA double-strand breaks, is highly overexpressed in glioblastoma. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that HJURP is remarkably overexpressed in a cohort composed of 40 patients with different grade astrocytomas. We also observed that tumors presenting the higher expression levels of HJURP are associated with poor survival prognosis, indicating HJURP overexpression as an independent prognostic factor of death risk for astrocytoma patients. More importantly, we found that HJURP knockdown strongly affects the maintenance of glioblastoma cells in a selective manner. Glioblastoma cells showed remarkable cell cycle arrest and premature senescence that culminated in elevated levels of cell death, differently from non-tumoral cells that were minimally affected. CONCLUSIONS: These data suggest that HJURP has an important role in the maintenance of extremely proliferative cells of high-grade gliomas and point to HJURP as a potential therapeutic target for the development of novel treatments for glioma patients.

Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.

Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions.

Melanoma survival strategies: the intrinsic apoptotic pathway: upstream and downstream regulators

Apoptotic cell death is involved in development and tissue homeostasis in numerous organisms, and changes in the apoptotic pathways are associated with many diseases, including cancer. The first evidence for an association between apoptosis and cancer was the discovery that the oncogene bcl-2 was involved in cell survival in lymphoma. Since then, alterations in the expression of genes that participate in cell survival pathways and resistance to apoptosis have become a hallmark of cancer. A failure to trigger apoptosis properly is an essential requirement during tumor progression and contributes to tumor resistance to radioor chemotherapy. Melanoma, one of the most aggressive cancers, is characterized by an elevated capacity to metastasize and by a high resistance to drugs. The strategies used by melanoma cells to avoid apoptosis often differ from those in other cancer cells. For example, in contrast to many tumors that frequently show a loss of p53 expression, melanoma maintains p53 expression but alters the p53 pathways. In this review, we summarize various aspects of melanocyte biology and consider the genetic alterations exploited by melanoma cells to escape apoptosis. We also discuss recent findings that have extended our understanding of the resistance of melanocytes to apoptosis during tumor progression.

Immunolocalization of myosin-V in the peribronchial, intrapulmonary peritracheal plexuses of the Wistar rat.

The location of myosin-V in whole mount preparations of the peritracheal and intrapulmonary peribronchial plexuses of Wistar rats has been shown by using an affinity-purified antibody specific to the medial tail domain of myosin-V. Myosin-V immunostaining was intense in the peritracheal and intrapulmonary peribronchial plexuses, allowing the visualization of neuronal cell bodies and fibers. Knowledge of the cellular localization and function of this class of myosin is an important achievement, as it allows the study of these plexuses so as to clarify the importance of the complex mechanism responsible for the functioning of the airways.

Expression of c-Met proto-oncogene in metastatic macrophage x melanoma fusion hybrids: implication of its possible role in MSH-induced motility.

It was shown previously that a majority of hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells displayed macrophage-specific glycosylation, especially increased GnT-V activity, beta1,6 branch formation in glycoproteins, accompanied by enhanced metastatic potential in vivo and motility in vitro. These hybrids also express upregulated melanocortin-1 receptor (MC1-R) activity and exhibit increased motility after melanocyte-stimulating hormone (MSH) treatment. In this report, we show that MSH-mediated stimulation of motility is mediated through enhanced expression of c-Met proto-oncogene. In metastatic hybrids c-Met expression is induced by MSH, and addition of c-Met neutralizing antibody to cells inhibits MSH-induced motility but not the basal motility of the cells. Furthermore, abrogation of the chemoattractant gradient concentration by addition of hepatocyte growth factor (HGF) recombinant protein, a cognate ligand of c-Met receptor, reduces the MSH-induced effect on motility. A similar result was also obtained by the addition of blocking anti-alphaHGF antibody in the chemoattractant chamber. Again, the metastatic hybrids, but not the nonmetastatic hybrids or parental melanoma cells, showed significant motile response to rHGF chemoattractant, and that motility is further induced when cells were stimulated with MSH/isobutylmethyl xanthine (IBMX). Synergistic stimulation on motility was also observed with those hybrids treated with MSH/IBMX and when rHGF and fibronectin (FN), in combination, were used as chemoattractants. These indicate that MSH/IBMX-induced motility might involve c-Met pathways as well as extracellular matrix (ECM)/integrin pathways in a cooperative fashion. Ets-1, a transcription factor involved in the expression of c-Met, is also found to be induced in metastatic hybrids after exposure to MSH/IBMX. Implication of the result is discussed in light of the role of c-Met and its interacting proteins in the development of metastatic phenotypes and its therapeutic intervention.

Expression of constructs of the neuronal isoform of myosin-Va interferes with the distribution of melanosomes and other vesicles in melanoma cells.

Myosin-Va has been implicated in melanosome translocation, but the exact molecular mechanisms underlying this function are not known. In the dilute, S91 melanoma cells, melanosomes move to the cell periphery but do not accumulate in the tips of dendrites as occurs in wild-type B16 melanocytes; rather, they return and accumulate primarily at the pericentrosomal region in a microtubule-dependent manner. Expression of the full-length neuronal isoform of myosin-Va in S91 cells causes melanosomes to disperse, occupying a cellular area approximately twice that observed in non-transfected cells, suggesting a partial rescue of the dilute phenotype. Overexpression of the full tail domain in S91 cells is not sufficient to induce melanosome dispersion, rather it causes melanosomal clumping. Overexpression of the head and head-neck domains of myosin-Va in B16 cells does not alter the melanosome distribution. However, overexpression of the full tail domain in these cells induces melanosome aggregation and the appearance of tail-associated, aggregated particles or vesicular structures that exhibit variable degrees of staining for melanosomal and Golgi beta-COP markers, as well as colocalization with the endogenous myosin-Va. Altogether, the present data suggest that myosin-Va plays a role in regulating the direction of microtubule-dependent melanosome translocation, in addition to promoting the capture of melanosomes at the cell periphery as suggested by previous studies. These studies also reinforce the notion that myosin-V has a broader function in melanocytes by acting on vesicular targeting or intracellular protein trafficking.