Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44%. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG) , and within the inhA promoter and/or in structural gene. A small percentage (~ 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh) . Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

The IFN-³+874T/A gene polymorphism is associated with retinochoroiditis toxoplasmosis susceptibility

Toxoplasmosis is a worldwide zoonosis that generally produces an asymptomatic infection. In some cases, however, toxoplasmosis infection can lead to ocular damage. The immune system has a crucial role in both the course of the infection and in the evolution of toxoplasmosis disease. In particular, IFN-³ plays an important role in resistance to toxoplasmosis. Polymorphisms in genes encoding cytokines have been shown to have an association with susceptibility to parasitic diseases. The aim of this work was to analyse the occurrence of polymorphisms in the gene encoding IFN-³ (+874T/A) among Toxoplasma gondii seropositive individuals, including those with ocular lesions caused by the parasite, from a rural population of Santa Rita de Cássia, Barra Mansa, state of Rio de Janeiro, Brazil. Further, we verified which of these polymorphisms could be related to susceptibility to the development of ocular toxoplasmosis. This study included 34 individuals with ocular toxoplasmosis (ocular group) and 134 without ocular lesions (control group). The differences between A and T allele distributions were not statistically significant between the two groups. However, we observed that a higher frequency of individuals from the ocular group possessed the A/A genotype, when compared with the control group, suggesting that homozygocity for the A allele could enhance susceptibility to ocular toxoplasmosis in T. gondii infection.

Genetic characterisation of Langerin gene in human immunodeficiency virus-1-infected women from Bahia, Brazil

Studies on human genetic variations are a useful source of knowledge about human immunodeficiency virus (HIV)-1 infection. The Langerin protein, found at the surface of Langerhans cells, has an important protective role in HIV-1 infection. Differences in Langerin function due to host genetic factors could influence susceptibility to HIV-1 infection. To verify the frequency of mutations in the Langerin gene, 118 samples from HIV-1-infected women and 99 samples from HIV-1-uninfected individuals were selected for sequencing of the promoter and carbohydrate recognition domain (CRD)-encoding regions of the Langerin gene. Langerin promoter analysis revealed two single nucleotide polymorphisms (SNPs) and one mutation in both studied groups, which created new binding sites for certain transcription factors, such as NFAT5, HOXB9.01 and STAT6.01, according to MatInspector software analysis. Three SNPs were observed in the CRD-encoding region in HIV-1-infected and uninfected individuals: p.K313I, c.941C>T and c.983C>T. This study shows that mutations in the Langerin gene are present in the analysed populations at different genotypic and allelic frequencies. Further studies should be conducted to verify the role of these mutations in HIV-1 susceptibility.

Low-P tolerance mechanisms and differential gene expression in contrasting wheat genotypes

The objectives of this study were to determine low-P tolerance mechanisms in contrasting wheat genotypes and to evaluate the association of these mechanisms to differential gene expression. Wheat seedlings of cultivars Toropi (tolerant to low-P availability) and Anahuac (sensitive) were evaluated. Seedlings were hydroponically grown in the absence or presence of P (1.0 mmol L-1) during three different time periods: 24, 120 and 240 hours. Free phosphate (Pi) and total P contents were measured in shoots and roots. The experiment's design was in randomized blocks with three replicates, each formed by ten plants. The relative expression of genes encoding the malate transporter TaALMT1 and the transcription factor PTF1 was evaluated. Phosphorus starvation beyond ten days increased the expression of TaALMT1 only in 'Toropi'. PTF1's expression was early induced in both genotypes under P starvation, but remained significant after ten days only in 'Toropi'. Shoot Pi concentration in 'Toropi' was independent from P availability; under starvation, 'Toropi' favored the maintenance of shoot Pi concentration. The low-P tolerance of Toropi cultivar at initial growth stages is mainly due to its ability to maintain constant the Pi shoot level.

Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods

Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR) and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT). The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

Occurrence of enterotoxin-encoding genes in Staphylococcus aureus causing mastitis in lactating goats

Staphylococcal enterotoxins are the leading cause of human food poisoning worldwide. Staphylococcus spp. are the main mastitis-causing agents in goats and frequently found in high counts in goat milk. This study aimed to investigate the occurrence of enterotoxin-encoding genes in Staphylococcus aureus associated with mastitis in lactating goats in Paraiba State, Brazil. Milk samples (n=2024) were collected from 393 farms. Staphylococcus aureus was isolated in 55 milk samples. Classical (sea, seb, sec, sed, see) and novel (seg, seh, sei) enterotoxin-encoding genes were investigated by means of polymerase chain reaction (PCR). From thirty-six tested isolates, enterotoxin-encoding genes were detected in 7 (19.5%) S. aureus. The gene encoding enterotoxin C (seC) was identified in six isolates, while seiwas observed in only one isolate. The genes sea, seb, sed, see, seg and seh were not observed amongst the S. aureus investigated in this study. In summary, S. aureus causing mastitis in goats can harbor enterotoxin-encoding genes and seC was the most frequent gene observed amongst the investigated isolates. This finding is important for surveillance purposes, since enterotoxin C should be investigated in human staphylococcal food poisoning outbreaks caused by consumption of goat milk and dairy products.

Oncogene-mediated downregulation of RECK, a novel transformation suppressor gene

The RECK gene was initially isolated as a transformation suppressor gene encoding a novel membrane-anchored glycoprotein and later found to suppress tumor invasion and metastasis by regulating matrix metalloproteinase-9. Its expression is ubiquitous in normal tissues, but undetectable in many tumor cell lines and in fibroblastic lines transformed by various oncogenes. The RECK gene promoter has been cloned and characterized. One of the elements responsible for the oncogene-mediated downregulation of mouse RECK gene is the Sp1 site, where the Sp1 and Sp3 factors bind. Sp1 transcription factor family is involved in the basal level of promoter activity of many genes, as well as in dynamic regulation of gene expression; in a majority of cases as a positive regulator, or, as exemplified by the oncogene-mediated suppression of RECK gene expression, as a negative transcription regulator. The molecular mechanisms of the downregulation of mouse RECK gene and other tumor suppressor genes are just beginning to be uncovered. Understanding the regulation of these genes may help to develop strategies to restore their expression in tumor cells and, hence, suppress the cells' malignant behavior.

ß-Spectrin São PauloII, a novel frameshift mutation of the ß-spectrin gene associated with hereditary spherocytosis and instability of the mutant mRNA

Hereditary spherocytosis (HS) is a common inherited anemia characterized by the presence of spherocytic red cells. Defects in several membrane protein genes have been involved in the pathogenesis of HS. ß-Spectrin-related HS seems to be common. We report here a new mutation in the ß-spectrin gene coding region in a patient with hereditary spherocytosis. The patient presented acanthocytosis and spectrin deficiency and, at the DNA level, a novel frameshift mutation leading to HS, i.e., a C deletion at codon 1392 (ß-spectrin São PauloII), exon 20. The mRNA encoding ß-spectrin São PauloII was very unstable and the mutant protein was not detected in the membrane or in other cellular compartments. It is interesting to note that frameshift mutations of the ß-spectrin gene at the 3' end allow the insertion of the mutant protein in the red cell membrane, leading to a defect in the auto-association of the spectrin dimers and consequent elliptocytosis. On the other hand, ß-spectrin São PauloII protein was absent in the red cell membrane, leading to spectrin deficiency, HS and the presence of acanthocytes.

Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

A novel missense mutation p.L76P in the GJB2 gene causing nonsyndromic recessive deafness in a Brazilian family

Mutations in the GJB2 gene, encoding connexin 26 (Cx26), are a major cause of nonsyndromic recessive hearing loss in many countries. We report here on a novel point mutation in GJB2, p.L76P (c.227C>T), in compound heterozygosity with a c.35delG mutation, in two Brazilian sibs, one presenting mild and the other profound nonsyndromic neurosensorial hearing impairment. Their father, who carried a wild-type allele and a p.L76P mutation, had normal hearing. The mutation leads to the substitution of leucine (L) by proline (P) at residue 76, an evolutionarily conserved position in Cx26 as well as in other connexins. This mutation is predicted to affect the first extracellular domain (EC1) or the second transmembrane domain (TM2). EC1 is important for connexon-connexon interaction and for the control of channel voltage gating. The segregation of the c.227C>T (p.L76P) mutation together with c.35delG in this family indicates a recessive mode of inheritance. The association between the p.L76P mutation and hearing impairment is further supported by its absence in a normal hearing control group of 100 individuals, 50 European-Brazilians and 50 African-Brazilians.

Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Gene expression of estrogen and oxytocin receptors in the uterus of pregnant and parturient bitches

In the canine species, the precise mechanisms of pregnancy maintenance and the initiation of parturition are not completely understood. The expression of genes encoding the receptors for estrogen (ERα mRNA) and oxytocin (OTR mRNA) was studied in the endometrium and myometrium during pregnancy and parturition in dogs. Real-time PCR was performed to quantify the levels of ERα mRNA and OTR mRNA in the uterus of bitches during early (up to 20 days of gestation), mid (20 to 40 days) and late pregnancy (41 to 60 days), and parturition (first stage of labor). All tissues expressed ERα and OTR mRNA, and are thus possibly able to respond to eventual estrogen and oxytocin hormonal stimuli. No statistically significant differences in the expression of ERα mRNA were verified in the endometrium and myometrium throughout pregnancy and parturition, but expression of OTR mRNA increased at both parturition and late pregnancy. We concluded that the increase of endometrial and myometrial OTR mRNA expression in dogs is not an event dependent on estrogenic stimulation. Moreover, the contractility response of the canine uterus to oxytocin begins during pregnancy and maintains myometrial activity. The expression of OTR mRNA in canine uterine tissues varied over time, which supports an interpretation that the sensitivity and response to hormone therapy varies during the course of pregnancy and labor. Further studies are needed to elucidate the factors underlying the synthesis of uterine oxytocin receptors and the possible role of ERβ rather than ERα in the uterine tissues during pregnancy and parturition in dogs.

Gene expression profiling analysis of lung adenocarcinoma

The present study screened potential genes related to lung adenocarcinoma, with the aim of further understanding disease pathogenesis. The GSE2514 dataset including 20 lung adenocarcinoma and 19 adjacent normal tissue samples from 10 patients with lung adenocarcinoma aged 45-73 years was downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) between the two groups were screened using the t-test. Potential gene functions were predicted using functional and pathway enrichment analysis, and protein-protein interaction (PPI) networks obtained from the STRING database were constructed with Cytoscape. Module analysis of PPI networks was performed through MCODE in Cytoscape. In total, 535 upregulated and 465 downregulated DEGs were identified. These included ATP5D, UQCRC2, UQCR11 and genes encoding nicotinamide adenine dinucleotide (NADH), which are mainly associated with mitochondrial ATP synthesis coupled electron transport, and which were enriched in the oxidative phosphorylation pathway. Other DEGs were associated with DNA replication (PRIM1, MCM3, and RNASEH2A), cell surface receptor-linked signal transduction and the enzyme-linked receptor protein signaling pathway (MAPK1, STAT3, RAF1, and JAK1), and regulation of the cytoskeleton and phosphatidylinositol signaling system (PIP5K1B, PIP5K1C, and PIP4K2B). Our findings suggest that DEGs encoding subunits of NADH, PRIM1, MCM3, MAPK1, STAT3, RAF1, and JAK1 might be associated with the development of lung adenocarcinoma.