Differential expression of Egr-1-like DNA-binding activities in the naive rat brain and after excitatory stimulation

Egr-1 and related proteins are inducible transcription factors within the brain recognizing the same consensus DNA sequence. Three Egr DNA-binding activities were observed in regions of the naive rat brain. Egr-1 was present in all brain regions examined. Bands composed, at least in part, of Egr-2 and Egr-3 were present in different relative amounts in the cerebral cortex, striatum, hippocampus, thalamus, and midbrain. All had similar affinity and specificity for the Egr consensus DNA recognition sequence. Administration of the convulsants NMDA, kainate, and pentylenetetrazole differentially induced Egr-1 and Egr-2/3 DNA-binding activities in the cerebral cortex, hippocampus, and cerebellum. All convulsants induced Egr-1 and Egr-2 immunoreactivity in the cerebral cortex and hippocampus. These data indicate that the members of the Egr family are regulated at different levels and may interact at promoters containing the Egr consensus sequence to fine tune a program of gene expression resulting from excitatory stimuli.

Evaluation of the insulin-like growth factors (IGF) IGF-I and IGF binding protein 3 in patients at high risk for breast cancer

Our data suggest that serum concentrations of insulin-like growth factor I and insulin-like growth factor binding protein 3 do not correlate with breast cancer development. (Fertil Steril (R) 2011;95:2753-5. (C)2011 by American Society for Reproductive Medicine.)

COL1A1 Sp1-binding site polymorphism as a risk factor for genital prolapse

The objective of this study was to verify the possible association between the Sp1-binding site polymorphism and genital prolapse. A case-control study was conducted in 107 patients with stages III and IV genital prolapse. The control group included 209 women with stages 0 and I. The polymorphism of type I collagen Sp1-binding site was identified by amplification of the first intron of the COL1A1 gene. We did not find differences in the prevalence of the GT and TT genotypes between the groups (p=0.34), even when we grouped patients with at least one polymorphic allele (GT and TT) and compared them with patients without the polymorphic allele (GG; p=0.17) The presence of at least one vaginal delivery, family history for prolapse, and macrosomatic fetus were independent risk factors for prolapse. In conclusion, the COL1A1 Sp1-binding site was not significantly associated with genital prolapse among our study subjects.

Penile erection induced in vivo by a purified toxin from the Brazilian spider Phoneutria nigriventer

OBJECTIVES To verify the effect on erectile tissue of mice of two neuropeptides extracted from the poison of a spider, Phoneutric nigriventer, (Tx2-5 and -6. termed `eretina`) after direct injection into the corpus cavernosum, to assess the minimum dosage necessary for effect. the time for initiation of action, the local duration of the erection, histological effects and the presence of local and systemic side-effects. MATERIALS AND METHODS When applied intraperitoneally, eretina promotes the relaxation of cavernous smooth muscle, thus causing penile erection. Thirty-five mice were divided in two groups; 10 control mice were injected 20 mu L of saline solution, and in the treated group, 25 mice were divided into groups of five and each subgroup received eretina in decreasing doses (0.024, 0.012, 0.006, 0.003 and 0.0015 mu g/kg) until the minimum dose that produced an erection was determined. After treatment 211 mice were monitored to determine the response and any collateral effects. RESULTS The minimum dose producing an erection was 0.006 mu g/kg, the five mice in this group having evidence of an erection at 35-45 min after injection. The histology of the cavernosum of mice treated with eretina showed dilatation and congestion of the vascular spaces with more blood than in controls. With the minimum dose there were no local or systemic collateral effects and the erection was lost after 120-140 min. CONCLUSION The minimum dose of eretina producing an erection in mice was determined. and the agent was safe for this use as it did not produce any collateral toxic effects. These studies indicate a possible means of determining the mechanism of action of eretina.

Mango starch degradation. II. The binding of alpha-amylase and beta-amylase to the starch granule

During mango ripening, soluble sugars that account for mango sweetening are accumulated through carbon supplied by both photosynthesis and starch degradation. The cultivar Keitt has a characteristic dependence on sugar accumulation during starch degradation, which takes place during ripening, only a few days after detachment from the tree. Most knowledge about starch degradation is based on seeds and leaves currently used as models. However, information about the mango fruit is scarce. This work presents the evaluation of alpha- and beta-amylases in the starch granule surface during fruit development and ripening. Extractable proteins were assayed for amylase activity and detected by immunofluorescence microscopy and correlated to gene expression. The results suggest that both amylases are involved in starch degradation during mango ripening, probably under the dependence of another signal triggered by the detachment from the mother-plant.

Moving ear syndrome: The role of botulinum toxin

We report a 30-year-old man with moving ear syndrome caused by focal myoclonic jerks of the right temporal muscle. This focal myoclonus would disappear while the patient was sleeping, swallowing, or speaking. He was treated with botulinum toxin type A with a favorable outcome. Previous reports of this condition and possible therapeutic approaches are discussed. (c) 2007 Movement Disorder Society.

Angiotensin II Binding to Angiotensin I-Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system. (Hypertension. 2011;57:965-972.) . Online Data Supplement

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. (C) 2009 Elsevier B.V. All rights reserved.

Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization

Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. it is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 min after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells. (C) 2008 Elsevier Ltd. All rights reserved.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B-1 and B-2 (kinin receptors) and AT(1) and AT(2) (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B, agonists Des-Arg(9)-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg(9)-BK or Des-Arg(10)-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT, agonist (Asp-Arg-Val-Tyr-lle-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B-1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT, receptor. To investigate this hypothesis, we replaced Arg(212) for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B, receptor by the 32 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg(212) in the TM V and a region of TM VI of rat B, receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII. (c) 2007 Elsevier B.V. All rights reserved.