Challenges in recombinant brain-derived neurotrophic factor production.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin of marked commercial, scientific, diagnostic, and therapeutic interest. The preservation of its structural cystine-knot is the main challenge in its industrial production. A suitable expression system is critical to achieve the most efficient production of bioactive and stable BDNF for pharmaceutical purposes.

Antiproliferative and pro-apoptotic activities of 2'- and 4'-aminochalcones against tumor canine cells.

In the present study, a series of 2'- and 4'-aminochalcones were synthesized and their antiproliferative activity against a canine malignant histiocytic cell line (DH82) was evaluated. Particularly aminochalcones with a hydrophobic substituent on ring B proved to be potent antiproliferative agents. Among these compounds, aminochalcones 3, 4 and 11 inhibited the growth of DH82 cells, with IC50 values of 34.4, 31.4 and 38.2 µM, respectively, and were three times more potent than etoposide (IC50 = 95.5 µM). The selected chalcones induced death through apoptosis rather than necrosis in DH82 and non-tumorigenic Madin-Darby canine kidney cells (MDCK). Further experiments suggested that the aminochalcones interfere with the regulation of oncogenes/tumor suppressor genes. Aminochalcone 11 inhibited transcription of the TOPOIIα and TP53 genes and aminochalcone 4 down-regulated Sp1 protein expression in a concentration-dependent manner.

Thymus vulgaris L. essential oil and its main component thymol: Anthelmintic effects against Haemonchus contortus from sheep.

Haemonchus contortus is an important gastrointestinal parasite on sheep farms in tropical regions. The resistance of the parasite against most anthelmintic drugs represents a great economic problem to sheep farming and is a major challenge that needs to be overcome. The searches for new anthelmintic agents that act on different stages of the parasite's life cycle are necessary for the development of new therapeutic options. The aim of this study was to evaluate the in vitro and in vivo anthelmintic activity of Thymus vulgaris essential oil against H. contortus and of its main component, the monoterpene thymol. Despite the relative ineffectiveness of the oil in the in vivo test, which may be corrected in the future after technical improvements to increase the oil's bioavailability, the in vitro results validated the popular use of T. vulgaris oil as an anthelmintic agent, at least against H. contortus. In fact, both the essential oil and thymol, which accounts for 50.22% of the oil composition, were effective against the three main stages of H. contortus. The oil and thymol were able to inhibit egg hatching by 96.4-100%, larval development by 90.8-100%, and larval motility by 97-100%. Similar to the positive control (levamisole 20mg/mL), the oil and thymol completely inhibited the motility of H. contortus adults within the first 8h of the experiment. Since thymol reproduces the anthelmintic effects of the oil and because it is the main component of the oil, it is reasonable to assume that thymol is the most important compound responsible for the anthelmintic effect of T. vulgaris. These results are of ethnopharmacological importance and may contribute to the development of new drugs and even herbal medicines, increasing treatment options for the farm breeding.

Typical Monoterpenes as Insecticides and Repellents against Stored Grain Pests.

Five monoterpenes naturally occurring in essential oils were tested for their insecticidal and repellent activities against the bruchid beetle Callosobruchus maculatus and the maize weevil Sitophilus zeamais. The monoterpenes were highly efficient as inducers of mortality or repellency against both insect species. They were more efficient in their fumigant activity against C. maculatus than against S. zeamais, while this profile of action was inverted when considering the repellent activities. Eugenol was one the most effective fumigants against both insects and one the most effective repellent against C. maculatus, while citronellal and geranial were one the most effective repellents against S. zeamais. Functional and positional isomerism of the monoterpenes pairs appears to exert little or no influence on theirs effects, especially in case of repellency. The validation of the insecticidal/repellent efficacy of isolated monoterpenes may permit a more advantageous, rapid, economic and optimized approach to the identification of promising oils for commercial formulations when combined with ethnobotanical strategies.

Antifungal activity of metabolites from the marine sponges Amphimedon sp. and Monanchora arbuscula against Aspergillus flavus strains isolated from peanuts (Arachis hypogaea).

Contamination of preharvest and stored peanuts (Arachis hypogaea L.) by aflatoxigenic strains of Aspergillus flavus is an important economical and food safety problem in many tropical and subtropical areas of the world. The present investigation reports the antifungal activity of a halitoxins/amphitoxins enriched extract obtained from the sponge Amphimedon sp. (HAEEAsp), and of batzelladine L isolated from the sponge Monanchora arbuscula on Aspergillus flavus isolated from stored peanuts. A PCR system directed against the ITS region and aflatoxin biosynthetic pathway genes of A. flavus was applied for identification of aflatoxin producing strains. The HAEEAsp extract and batzelladine L showed minimal inhibitory concentration (MIC) in the range between 1.9 to 15.6 microg/mL and between 1.9 to 7.8 microg/mL, respectively. The minimal fungicide concentration (MFC) of HAEEAsp extract and batzelladine L was in the range between 3.9 to 31.3 microg/mL and 3.9 to 15.6 microg/mL, respectively. These results indicate that these marine alkaloids may be further explored for the development of potential lead compounds active against aflatoxigenic fungi.

Delayed effects of exposure to a moderate radiation dose on transcription profiles in human primary fibroblasts.

Ionizing radiation (IR) is used in a wide variety of medical and nonmedical applications and poses a potential threat to human health. Knowledge of changes in gene expression in irradiated cells may be helpful for the establishment of effective paradigms for radiation protection. IR-induced DNA damage triggers a complex cascade of signal transduction. Recently, genome-wide approaches have allowed the detection of alterations in gene expression across a wide range of radiation doses. However, the delayed or long-term biological effects of mild-doses of IR remain largely unknown. The main objective of the present study was to investigate the effects of a moderate dose of gamma-rays (50 cGy) on gene expression 6 days post-irradiation. Gene expression using cDNA microarrays revealed statistically significant changes in the expression of 59 genes (FDR < 0.07), whose functions are related to cell-cycle control, protein trafficking, ubiquitin cycle, Rho-GTPAse pathway, protein phosphatase signalization, oxidoreductase control, and stress response. A set of 464 genes was also selected by a less stringent approach, and we demonstrate that this broader set of genes can efficiently distinguish the irradiated samples from the unirradiated, defining a long-term IR signature in human primary fibroblasts. Our findings support the existence of persistent responses to mild doses of IR detectable by changes in gene expression profiles. These results provide insight into delayed effects observed in human primary cells as well as the role of long-term response in neoplastic transformation. Environ.

Shared and unique gene expression in systemic lupus erythematosus depending on disease activity.

Patients presenting with active systemic lupus erythematosus (SLE) manifestations may exhibit distinct pathogenetic features in relation to inactive SLE. Also, cDNA microarrays may potentially discriminate the gene expression profile of a disease or disease variant. Therefore, we evaluated the expression profile of 4500 genes in peripheral blood lymphocytes (PBL) of SLE patients. We studied 11 patients with SLE (seven with active SLE and four with inactive SLE) and eight healthy controls. Total RNA was isolated from PBL, reverse transcribed into cDNA, and postlabeled with Cy3 fluorochrome. These probes were then hybridized to a glass slide cDNA microarray containing 4500 human IMAGE cDNA target sequences. An equimolar amount of total RNA from human cell lines served as reference. The microarray images were quantified, normalized, and analyzed using the R environment (ANOVA, significant analysis of microarrays, and cluster-tree view algorithms). Disease activity was assessed by the SLE disease activity index. Compared to the healthy controls, 104 genes in active SLE patients (80 repressed and 24 induced) and 52 genes in nonactive SLE patients (31 induced and 21 repressed) were differentially expressed. The modulation of 12 genes, either induced or repressed, was found in both disease variants; however, each disease variant had differential expression of different genes. Taken together, these results indicate that the two lupus variants studied have common and unique differentially expressed genes. Although the biological significance of the differentially expressed genes discussed above has not been completely understood, they may serve as a platform to further explore the molecular basis of immune deregulation in SLE.

Transcriptional response of murine macrophages upon infection with opsonized Paracoccidioides brasiliensis yeast cells.

Paracoccidioides brasiliensis is the etiologic agent of the Paracoccidioidomycosis the most common systemic mycosis in Latin America. Little is known about the regulation of genes involved in the innate immune host response to P. brasiliensis. We therefore examined the kinetic profile of gene expression of peritoneal macrophage infected with P. brasiliensis. Total RNA from macrophages at 6, 24 and 48h was extracted, hybridized onto nylon membranes and analyzed. An increase in the transcription of a number of pro-inflammatory molecules encoding membrane proteins, metalloproteases, involved in adhesion and phagocytosis, are described. We observed also the differential expression of genes whose products may cause apoptotic events induced at 24h. In addition, considering the simultaneous analyses of differential gene expression for the pathogen reported before by our group, at six hours post infection, we propose a model at molecular level for the P. brasiliensis-macrophage early interaction. In this regard, P. brasiliensis regulates genes specially related to stress and macrophages, at the same time point, up-regulate genes related to inflammation and phagocytosis, probably as an effort to counteract infection by the fungus.

Gene expression profiles in human lymphocytes irradiated in vitro with low doses of gamma rays.

The molecular mechanisms underlying responses to low radiation doses are still unknown, especially in normal lymphocytes, despite the evidence suggesting specific changes that may characterize cellular responses. Our purpose was to analyze gene expression profiles by DNA microarrays in human lymphocytes after in vitro irradiation (10, 25 and 50 cGy) with gamma rays. A cytogenetic analysis was also carried out for different radiation doses. G 0 lymphocytes were irradiated and induced to proliferate for 48 h; then RNA samples were collected for gene expression analysis. ANOVA was applied to data obtained in four experiments with four healthy donors, followed by SAM analysis and hierarchical clustering. For 10, 25 and 50 cGy, the numbers of significantly (FDR or=10 cGy (total aberrations) and >or=50 cGy (dicentrics/ rings). Therefore, low to moderate radiation doses induced qualitative and/or quantitative differences and similarities in transcript profiles, reflecting the type and extent of DNA lesions. The main biological processes associated with modulated genes were metabolism, stress response/DNA repair, cell growth/differentiation, and transcription regulation. The results indicate a potential risk to humans regarding the development of genetic instability and acquired diseases.

Early transcriptional response of Paracoccidioides brasiliensis upon internalization by murine macrophages.

Paracoccidioides brasiliensis, a thermal dimorphic fungus, is the etiologic agent of the most common systemic mycosis in Latin America, paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen being able to survive and replicate within the phagosome of nonactivated murine and human macrophages. This ability has been proposed to be crucial to the development of disease. Thus, P. brasiliensis may have evolved mechanisms that counteract the constraints imposed by phagocytic cells. By using cDNA microarray technology we evaluated the early transcriptional response of this fungus to the environment of peritoneal murine macrophages in order to shed light on the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genes analyzed, we identified 152 genes that were differentially transcribed. Intracellularly expressed genes were primarily associated with glucose and amino acid limitation, cell wall construction, and oxidative stress. For the first time, a comprehensive gene expression tool is used for the expression analysis of P. brasiliensis genes when interacting with macrophages. Overall, our data show a transcriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages which may lead to adaptation and consequent survival of this pathogen.

Is HLA class II profile relevant for the study of large-scale differentially expressed genes in type 1 diabetes mellitus patients?

We have previously identified 30 differentially expressed genes when comparing recently diagnosed type 1 diabetes mellitus (DM-1) patients and controls paired for sex, age, and ethnic background. In this article we performed the hierarchical clustering of these genes taking into account the human-leukocyte-antigen (HLA)-DRB1/DQB1 profile. The dendrogram obtained using the Cluster program grouped patients and controls into three clusters, one including individuals with no susceptibility alleles, another including individuals with at least three susceptibility alleles, and a third intermingling susceptibility/protective alleles. In addition to other variables, the results of the present article suggest that the major histocompatibility complex (MHC) class II profile may be of relevance for the study of a large-scale differentially expressed genes.

The pH signaling transcription factor PacC mediates the growth of Trichophyton rubrum on human nail in vitro.

We report here the isolation, molecular cloning and initial characterization of the Trichophytonrubrum pacC gene, which encodes a putative protein that is homologous to the PacC/Rim101p family of pH signaling transcription regulators. The promoter region of the T. rubrumpacC gene contains four recognition sites 5'-GCCAAG-3' for the PacC protein, suggesting that the transcription of this gene itself could be induced under alkaline growth conditions. The enhanced expression profile of the T. rubrumpacC gene in an alkaline environment was confirmed by Northern blotting analysis. We also report that the disruption of pacC gene decreased both the secretion of keratinolytic proteases and the ability of the mutant pacC-1 to grow on human nail fragments as the sole source of nutrition, i.e., growth of the dermatophyte T. rubrum appear to be related to molecular events which depend on the action of protein PacC.

Using cDNA microarrays to identify human CD19(+) B cell gene products (ESTs) originated from systemic lupus erythematosus susceptibility loci.

Systemic lupus erythematosus (SLE) is a prototype of autoimmune disease which arises from interactions between susceptibility genes and environmental factors. Despite the heterogeneous manifestations in this disease, all SLE patients present plasma autoantibodies recognizing nuclear components. Thus, auto reactive B cells represent key effectors to be investigated. Human linkage analysis is providing the localization of susceptibility loci distributed in chromosomes contributing to elucidate the manner in which interactions between these loci mediate SLE pathogenesis. We associate the cDNA microarray technology to investigate the differential gene expression of CD19(+) B cells with genetic linkage data. Bioinformatics programs served to evidentiate the differentially expressed sequences and the design of the microarray allowed hierarchical clustering of patients and controls. Sequencing allowed the identification of 8 new gene products differentially expressed (ESTs) that were co-localized in SLE or other autoimmune diseases susceptibility loci on chromosome 1p21, 2q21, 13q33, 16p12.1 and 16q12.1. These findings strongly suggest that chromosomal regions previously identified as SLE susceptibility loci are in fact transcribed in CD19(+) B cells of patients. In this review, we delineate a new possibility for the use of cDNA microarrays in studies focusing the control of gene expression of disease susceptibility loci identified by genetic linkage.

Transcriptional profiles of the human pathogenic fungus Paracoccidioides brasiliensis in mycelium and yeast cells.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Gene expression profiles in human cells submitted to genotoxic stress.

Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). In this report, we present three approaches to document gene expression profiles, dealing with the evaluation of cellular responses to genotoxic agents (gamma-rays from 60Cobalt and cyclophosphamide). We used the method of cDNA arrays to analyze the differential gene expression profiles that were displayed by lymphocytes from radiation-exposed individuals, a human fibroblast cell line, and T lymphocytes from systemic lupus erythematosus (SLE) patients who were treated with cyclophosphamide. A preliminary analysis performed in lymphocytes from three radiation-workers showed that several induced genes can be associated with cell response to ionizing radiation: TRRAP (cell cycle regulation), Ligase IV (DNA repair), MAPK8IP1 and MAPK10 (signal transduction), RASSF2 (apoptosis induction/tumorigenesis), p53 (damage response/maintenance of genetic stability). The in vitro irradiated normal VH16 cell line (primary) showed a complex response to the genotoxic stress at the molecular level. Many apoptotic pathways were concomitantly induced. In addition, several genes involved in signaling and cell cycle arrest/control were significantly modulated after irradiation. Many genes involved in oxidative damage were also induced, indicating that this mechanism seems to be an important component of cell response. After treatment of the SLE patients with cyclophosphamide, 154 genes were differentially and significantly induced. Among them, we identified those associated with drug detoxification, cell cycle control, apoptosis, and tumor-suppressor. These findings indicate that at least two apoptotic pathways were induced after cyclophosphamide treatment. The induction of APAF1 and two genes coding for two subunits of cytochrome c supports a previous report showing increased apoptosis in lymphocytes from SLE patients. The present study provides new information on the molecular mechanism underlying the cell response to genotoxic stress, with relevance to basic and clinical research.