The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5`-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere

Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

In vivo effects of TGF beta 1 on the the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGF beta) wanes in the milk of lactating rat, an increase in TGF beta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGF beta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGF beta 1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGF beta 1 while T beta RII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p < 0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p < 0.05). Importantly, TGF beta 1 inhibited cell proliferation (p < 0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p < 0.05). Also, TGF beta 1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p < 0.001), and by morphological features at 12 h (p < 0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGF beta 1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TG beta 1 in gastric growth during postnatal development, (C) 2007 Elsevier B.V. All rights reserved.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B(2) and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (< EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B(2) receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B(2) receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development. (C) 2011 Elsevier Inc. All rights reserved.

The Immunomodulatory Action of Sialostatin L on Dendritic Cells Reveals Its Potential to Interfere with Autoimmunity

Sialostatin L (SialoL) is a secreted cysteine protease inhibitor identified in the salivary glands of the Lyme disease vector Ixodes scapularis. In this study, we reveal the mechanisms of SialoL immunomodulatory actions on the vertebrate host. LPS-induced maturation of dendritic cells from C57BL/6 mice was significantly reduced in the presence of SialoL. Although OVA degradation was not affected by the presence of SialoL in dendritic cell cultures, cathepsin S activity was partially inhibited, leading to an accumulation of a 10-kDa invariant chain intermediate in these cells. As a consequence, in vitro Ag-specific CD4(+) T cell proliferation was inhibited in a time-dependent manner by SialoL, and further studies engaging cathepsin S(-/-) or cathepsin L(-/-) dendritic cells confirmed that the immunomodulatory actions of SialoL are mediated by inhibition of cathepsin S. Moreover, mice treated with SialoL displayed decreased early T cell expansion and recall response upon antigenic stimulation. Finally, SialoL administration during the immunization phase of experimental autoimmune encephalomyelitis in mice significantly prevented disease symptoms, which was associated with impaired IFN-gamma and IL-17 production and specific T cell proliferation. These results illuminate the dual mechanism by which a human disease vector protein modulates vertebrate host immunity and reveals its potential in prevention of an autoimmune disease. The Journal of Immunology, 2009, 182: 7422-7429.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito`s development. (C) 2010 Elsevier Masson SAS. All rights reserved.

Ethanol consumption and pineal melatonin daily profile in rats

It is well known that melatonin participates in the regulation of many important physiological functions such as sleep-wakefulness cycle, motor coordination and neural plasticity, and cognition. However, as there are contradictory results regarding the melatonin production diurnal profile under alcohol consumption, the aim of this paper was to study the phenomenology and mechanisms of the putative modifications on the daily profile of melatonin production in rats submitted to chronic alcohol intake. The present results show that rats receiving 10% ethanol in drinking water for 35 days display an altered daily profile of melatonin production, with a phase delay and a reduction in the nocturnal peak. This can be partially explained by a loss of the daily rhythm and the 25% reduction in tryptophan hydroxylase activity and, mainly, by a phase delay in arylalkylamine N-acetyltransferase gene expression and a 70% reduction in its peak activity. Upstream in the melatonin synthesis pathway, the results showed that noradrenergic signaling is impaired as well, with a decrease in beta 1 and alpha 1 adrenergic receptors` mRNA contents and in vitro sustained loss of noradrenergic-stimulated melatonin production by glands from alcohol-treated rats. Together, these results confirm the alterations in the daily melatonin profile of alcoholic rats and suggest the possible mechanisms for the observed melatonin synthesis modification.

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland

Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.

Sodium Tungstate on Some Biochemical Parameters of the Parotid Salivary Gland of Streptozotocin-Induced Diabetic Rats: A Short-Term Study

Several studies have shown the antidiabetic properties of sodium tungstate. In this study, we evaluated some biochemical parameters of the parotid salivary gland of streptozotocin-induced diabetic rats treated with sodium tungstate solution (2 mg/ml). The studied groups were: untreated control (UC), treated control (TC), untreated diabetic (UD), and treated diabetic (TD). After 2 and 6 weeks of treatment, parotid gland was removed and total protein and sialic acid (free and total) concentration and amylase and peroxidase activities were determined. Data were compared by variance analysis and Tukey test (p < 0.05). The sodium tungstate treatment modestly decreased the glycemia of streptozotocin-induced diabetic rats. At week 2 of the study, parotid gland of diabetic rats presented a reduction of total protein concentration (55%) and an increase of amylase (120%) and peroxidase (160%) activities, free (150%) and total (170%) sialic acid concentration. No alteration in the evaluated parameters at week 6 of the study was observed. Sodium tungstate presented no significant effect in parotid gland. Our results suggest that diabetes causes initial modification in biochemical composition of parotid. However, this gland showed a recovery capacity after 6 week of the experimental time. Sodium tungstate has no effect in peripheral tissues, such as salivary glands.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts

Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (<= 36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.