A Mox homeobox gene in the gastropod mollusc Haliotis rufescens is differentially expressed during larval morphogenesis and metamorphosis

We have isolated a homeobox-containing cDNA from the gastropod mollusc Haliotis rufescens that is most similar to members of the Mox homeobox gene class, The derived Haliotis homeodomain sequence is 85% identical to mouse and frog Mox-2 homeodomains and 88.9% identical to the partial cnidarian cnox5-Hm homeodomain. Quantitative reverse transcription-polymerase chain reaction analysis of mRNA accumulation reveals that this gene, called HruMox, is expressed in the larva, but not in the early embryo, Transcripts are most prevalent during larval morphogenesis from trochophore to veliger. There are also transient increases in transcript prevalence 1 and 3 days after the intitiation of metamorphosis from veliger to juvenile. The identification of a molluscan Mox homeobox gene that is more closely related to vertebrate genes than other protostome (e.g. Drosophila) genes suggests the Mox class of homeobox genes may consist of several different families that have been conserved through evolution, (C) 1997 Federation of European Biochemical Societies.

Lack of galectin-3 alters the balance of innate immune cytokines and confers resistance to Rhodococcus equi infection

Galectin-3 is a p-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3(-/-)) and their wild-type counterpart (gal3(+/+)) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3(+/+) mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1 beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3(+/+) macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1 beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1 beta production and thus affecting resistance to R. equi infection.

Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.

Genic expression of the uterine leiomyoma in reproductive-aged women after treatment with goserelin

Objective: To identify the genes presenting different expression in uterine leiomyomas after goserelin treatment. Design: Retrospective analyses of tissue obtained in a prospective clinical study. Setting: School of Medicine of the University of Sao Paulo. Patient(s): 30 nulliparous black women aged 20 to 45 years with symptoms of uterine leiomyoma, uterine volume over 300 mL, and surgical indications for myomectomy. Intervention(s): Fifteen patients were given a monthly dose of 3.6 mg of goserelin over 3 months before surgery (group A), and 15 patients underwent surgery without any previous treatment (group B). Five random samples from each group were analyzed using the microarray technique with the Affymetrix platform (GeneChip Rat Genome 230 2.0 Array). Main Outcome Measure(s): Quantification of transcript expression levels of uterine fibroids in patients treated or not treated with goserelin. Result(s): Of the total of 47,000 sequences that were analyzed, representing approximately 38,500 human genes already characterized, we found a differential expression of 174 genes. Of these, 70 were up-regulated (33 genes with known function) and 104 were down-regulated (65 genes with known function) in samples from group A (treated) when compared with group B (nontreated). Conclusion(s): The genic expression of uterine leiomyomas changes in women who have had goserelin treatment when compared with nontreated patients. (Fertil Steril (R) 2010; 94: 1072-7. (C) 2010 by American Society for Reproductive Medicine.)

Association of low sodium-iodide symporter messenger ribonucleic acid expression in malignant thyroid nodules with increased intracellular protein staining

Context: The expression of sodium iodide symporter (NIS) is required for iodide uptake in thyroid cells. Benign and malignant thyroid tumors have low iodide uptake. However, previous studies by RT-PCR or immunohistochemistry have shown divergent results of NIS expression in these nodules. Objective: The objective of the study was to investigate NIS mRNA transcript levels, compare with NIS and TSH receptor proteins expression, and localize the NIS protein in thyroid nodules samples and their surrounding nonnodular tissues (controls). Design: NIS mRNA levels, quantified by real-time RT-PCR, and NIS and TSH receptor proteins, evaluated by immunohistochemistry, were examined in surgical specimens of 12 benign and 13 malignant nodules and control samples. Results: When compared with controls, 83.3% of the benign and 100% of the malignant nodules had significantly lower NIS gene expression. Conversely, 66.7% of the benign and 100% of malignant nodules had stronger intracellular NIS immunostaining than controls. Low gene expression associated with strong intracellular immunostaining was most frequently detected in malignant (100%) than benign nodules (50%; P = 0.005). NIS protein was located at the basolateral membrane in 24% of the control samples, 8.3% of the benign, and 15.4% of the malignant nodules. The percentage of benign nodules with strong TSH receptor positivity (41.6%) was higher than malignant (7.7%). Conclusion: We confirmed that reduced NIS mRNA expression in thyroid malignant nodules is associated with strong intracellular protein staining and may be related to the inability of the NIS protein to migrate to the cellular basolateral membrane. These results may explain the low iodide uptake of malignant nodules.

Claudin-7 down-regulation is an important feature in oral squamous cell carcinoma

Aims: Claudins, a large family of essential tight junction (TJ) proteins, are abnormally regulated in human carcinomas, especially claudin-7. The aim of this study was to investigate claudin-7 expression and alterations in oral squamous cell carcinoma (OSCC). Methods and results: Expression of claudin-7 was analysed in 132 cases of OSCC organized in a tissue microarray. Claudin-7 mRNA transcript was evaluated using real-time polymerase chain reaction and the methylation status of the promoter was also assessed. Claudin-7 was negative in 58.3% of the cases. Loss of claudin-7 protein expression was associated with recurrence (P = 0.019), tumour size (P = 0.014), clinical stage of OSCC (P = 0.055) and disease-free survival (P = 0.015). Down-regulation of the claudin-7 mRNA transcripts was observed in 78% of the cases, in accordance with immunoexpression. Analysis of the methylation status of the promoter region of claudin-7 revealed that treatment of O28 cells (that did not express claudin-7 mRNA transcripts) with 5-Aza-2`-Deoxycytidine (5-Aza-dC) led to the re-expression of claudin-7 mRNA transcript. Conclusion: Loss of claudin-7 expression is associated with important subcellular processes in OSCC with impact on clinical parameters.

Differential display and suppression subtractive hybridization analysis of the pulp of ripening banana

The fruit of banana undergoes several important physico-chemical changes during ripening. Analysis of gene expression would permit identification of important genes and regulatory elements involved in this process. Therefore, transcript profiling of preclimacteric and climacteric fruit was performed using differential display and Suppression subtractive hybridization. Our analyses resulted in the isolation of 12 differentially expressed cDNAs, which were confirmed by dot-blots and northern blots. Among the sequences identified were sequences homologous to plant aquaporins, adenine nucleotide translocator, immunophilin, legumin-like proteins, deoxyguanosine kinase and omega-3 fatty acid desaturase. Some of these cDNAs correspond to newly isolated genes involved in changes related to the respiratory climacteric, or stress-defense responses. Functional characterization of ripening-associated genes could provide information useful in controlling biochemical pathways that would have an impact on banana quality and shelf life. (C) 2009 Elsevier B.V. All rights reserved.

Molecular cloning and characterization of a ripening-induced polygalacturonase related to papaya fruit softening

Pulp softening is one of the most remarkable changes during ripening of papaya (Carica papaya) fruit and it is a major cause for post-harvest losses. Although cell wall catabolism has a major influence on papaya fruit, quality information on the gene products involved in this process is limited. A full-length polygalacturonase cDNA (cpPG) was isolated from papaya pulp and used to study gene expression and enzyme activity during normal and ethylene-induced ripening and after exposure of the fruit to 1-MCP. Northern-blot analysis demonstrated that cpPG transcription was strongly induced during ripening and was highly ethylene-dependent. The accumulation of cpPG transcript was paralleled by enzyme activity, and inversely correlated to the pulp firmness. Preliminary in silica analysis of the cpPG genomic sequence revealed the occurrence of putative regulatory motifs in the promoter region that may help to explain the effects of plant hormones and non-abiotic stresses on papaya fruit firmness. This newly isolated cpPG is an important candidate for functional characterization and manipulation to control the process of pulp softening during papaya ripening. (C) 2009 Elsevier Masson SAS. All rights reserved.

Prognostic Impact of MYCN, DDX1, TrkA, and TrkC Gene Transcripts Expression in Neuroblastoma

Background. The aims of this study were to define the mRNA expression profiles of MYCN, DDX1, TrkA, and TrkC in biopsy tumor samples from 64 Brazilian patients with neuroblastomas of different risk stages and to correlate altered expression with prognostic values. Procedure. Patients were retrospectively classified into low- (n = 11), intermediate- (n = 18), and high-risk (n = 35) groups using standard criteria. The mRNA levels of the above genes were measured by quantitative real-time polymerase chain reaction. Univariate analyses were performed and survival curves were plotted by the Kaplan-Meier method. Results. Of the 64 patients, 53% were female and 62.5% were older than 18 months. The 5-year overall survival (OS) for the entire cohort was 40.3%, with inferior median OS in patients identified in the intermediate- and high-risk groups. A significant difference in OS with respect to TrkA mRNA expression was found for the high-risk group vs. either the low- or intermediate-risk groups (P < 0.01, log rank test). Within the intermediate-risk group, neuroblastoma patients with positive TrkA mRNA expression had better clinical outcomes than patients with no TrkA transcript expression (P = 0.004). Another difference in OS was only found between the intermediate- and high-risk groups (P < 0.027, log rank test). No significant correlation of mRNA expression and survival outcome could be detected for the MYCN, DDX1. Conclusions. Positive expression of TrkA mRNA may be a clinically useful addition to the current risk classification system, allowing the identification of NB tumors with favorable prognosis. Pediatr Blood Cancer 2011; 56: 749-756. (c) 2010 Wiley-Liss, Inc.

A catecholamine transporter from the human parasite Schistosoma mansoni with low affinity for psychostimulants

The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement. Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from S. mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercariae stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage. Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE. (C) 2011 Elsevier B.V. All rights reserved.

Evidences of a Role for Eukaryotic Translation Initiation Factor 5A (eIF5A) in Mouse Embryogenesis and Cell Differentiation

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation. J. Cell. Physiol. 225: 500-505, 2010. (C) 2010 Wiley-Liss, Inc.

Molecular cloning, sequencing, and expression analysis of presenilin cDNA from Schistosoma mansoni

Presenilins (PS) are integral membrane proteins involved, among other functions, in regulated intramembrane proteolysis. In this study, we report the identification and characterization of a complementary DNA from Schistosoma mansoni exhibiting a significant homology to human and nonvertebrate presinilins. S. mansoni contained a 1,485 bp open reading frame encoding a predicted protein of 494 amino acids. Alignment of predicted amino acid sequence of S. mansoni with PS (SmPS) from other species revealed up to 40% similarity shared among the investigated organisms. In addition, phylogenetic analyses demonstrated SmPS being closely related to its orthologues found in Schistosoma japonicum and Caenorhabditis elegans. Expression analysis of SmPS using quantitative real-time PCR revealed that the transcript is up-regulated in the egg stage. We hypothesize that the high level of SmPS in the S. mansoni embryo correlates to an important role during cellular signaling associated to larval development. To our knowledge, this study represents the first attempt to investigate the existence and abundance of PS from a helminth parasite.

Preliminary analysis of miRNA pathway in Schistosoma mansoni

RNA silencing refers to a series of nuclear and cytoplasmatic processes involved in the post-transcriptional regulation of gene expression or post-transcriptional gene silencing (PTGS), either by sequence-specific mRNA degradation or by translational at-rest. The best characterized small RNAs are microRNAs (miRNAs), which predominantly perform gene silencing through post-transcriptional mechanisms. in this work we used bioinformatic approaches to identify the parasitic trematode Schistosoma Mansoni sequences that are similar to enzymes involved in the post-transcriptional gene silencing mediated by miRNA pathway. We used amino acid sequences of well-known proteins involved in the miRNA pathway against S. mansoni genome and transcriptome databases identifying a total of 13 Putative proteins in the parasite. In addition, the transcript levels of SinDicer1 and SmAgo2/3/4 were identified by qRT-PCR using cercariae, adult worms, eggs and in vitro Cultivated schistosomula. Our results showed that the SmDicer1 and SmAgo2/3/4 are differentially expressed during schistosomula development, suggesting that the miRNA pathway is regulated at the transcript level and therefore may control gene expression during the life cycle of S. mansoni. (C) 2008 Published by Elsevier Ireland Ltd.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host. (c) 2010 Elsevier Ltd. All rights reserved.

The insulin signaling pathway in honey bee (Apis mellifera) caste development - differential expression of insulin-like peptides and insulin receptors in queen and worker larvae

The insulin/insulin-like signaling (IIS) pathway is an evolutionarily conserved module in the control of body size and correlated organ growth in metazoans. In the highly eusocial bees, the caste phenotypes differ not only in size and several structural features but also in individual fitness and life history. We investigated the developmental expression profiles of genes encoding the two insulin-like peptides (AmILP-1 and AmILP-2) and the two insulin receptors (AmInR-1 and AmInR-2) predicted in the honey bee genome. Quantitative PCR analysis for queen and worker larvae in critical stages of caste development showed that AmILP-2 is the predominantly transcribed ILP in both castes, with higher expression in workers than in queens. Expression of both InR genes sharply declined in fourth instar queen larvae, but showed little modulation in workers. On first sight, these findings are non-intuitive, considering the higher growth rates of queens, but they can be interpreted as possibly antagonistic crosstalk between the IIS module and juvenile hormone. Analyzing AmInR-1 and AmInR-2 expression in ovaries of queen and worker larvae revealed low transcript levels in queens and a sharp drop in AmInR-2 expression in fifth instar worker larvae, indicating relative independence in tissue-specific versus overall IIS pathway activity. (C) 2008 Elsevier Ltd. All rights reserved.