Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca²⁺ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.Financial support through grants and scholarships from the Brazilian funding agencies FAPESP, CNPq, and CAPES is gratefully acknowledged.     

Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.     

Effects of bound Ts3 on voltage dependence of sodium channel transitions to and from inactivation and energetics of its unbinding

Recently, we proposed a quantitative model to explain the molecular mechanism of action of the Tityus serrulatus Ts3 α-toxin on sodium channels. In this model, the toxin acts as a stop that prevents the segment S4 of domain IV from reaching its outermost position, thus impairing the normal fast inactivation without affecting activation. In the present work, we analyze the predictions of the proposed model with regard to the voltage-dependent transitions to and from inactivation. Our results show that the recovery from inactivation was significantly faster in Ts3-bound channels and that there was no significant voltage dependence. The transition to inactivated state from open state in Ts3-modified channels presented a small but significant voltage dependence, which may derive from an intrinsic voltage dependence of inactivation or by a short movement of IVS4 in the presence of bound Ts3. We also studied the thermodynamic parameters of the voltage-dependent displacement of Ts3 from its binding site. We have observed that the activation energy to remove the toxin is 27 kJ/mol, part of which derives from the imposed depolarizing potential and the movement of an equivalent electrical charge of 0.54 c ₀. These results support the proposed model.     

Description of nine South American species of the genus Neralsia cameron, with low scutellar carina (Hymenoptera: Cynipoidea: Figitidae)

In this study a total of nine new species of the genus Neralsia are described for South America: N. desantisi Jiménez & Pujade-Villar n. sp., N. equilatera Jiménez & Pujade-Villar n. sp., N. hermafrodita Jiménez & Pujade-Villar n. sp., N. marioi Jiménez & Pujade-Villar n. sp., N. moisesi Jiménez & Pujade-Villar n. sp., N. obelix Jiménez & Pujade-Villar n. sp., N. parafossulata Jiménez & Pujade-Villar n. sp., N. pseudoneralsia Jiménez & Pujade-Villar n. sp. and N. rauli Jiménez & Pujade-Villar n. sp., all of them have in common the low carina separating the scutellar foveae. Other six known Neralsia species, N. albipennis (Kieffer), N. bogotensis (Kieffer), N. flavidipennis (Kieffer), N. fossulata (Kieffer), N. pilosa (Borgmeier) and N. striaticeps (Kieffer) have the same scutellar character. The morphological characters to differentiate all mentioned species are presented.     

Cattle tick Boophilus microplus salivary gland contains a thiol-activated metalloendopeptidase displaying kininase activity

This work reports on the characterization of a metalloendopeptidase kininase present in Boophilus microplus salivary glands. Using the guinea pig ileum assay, salivary gland whole extracts (SGE) were found to have a potent kininase activity. Ion-exchange chromatography separated two kininase activities from SGE. The major enzymatic component, eluted at lower ionic strength, was named BooKase (Boophilus Kininase). Analysis of the hydrolysis products by capillary electrophoresis identified Phe5-Ser6 as the only hydrolyzable peptide bond in bradykinin after BooKase treatment. This is the same specificity as the mammalian thimet oligoendopeptidase (EC 3.4.24.15). Like this enzyme, BooKase is also a metallo-peptidase (requires Mn2+) and is activated by-SH protecting reagents. In addition, BooKase was partially inhibited by cFP-AAF-pAB, a specific inhibitor of thimet oligopeptidase. Contrary to other kininases, BooKase had no activity upon angiontensin I. Our results show that BooKase behaves as a typical peptidase with kinase activity.     

Nandrolone and stanozolol induce leydig cell tumor proliferation through an estrogen‐dependent mechanism involving IGF‐I system

Several substances such as anabolic androgenic steroids (AAS), peptide hormones like insulin‐like growth factor‐I (IGF‐I), aromatase inhibitors and estrogen antagonists are offered via the Internet, and are assumed without considering the potential deleterious effects that can be caused by their administration. In this study we aimed to determine if nandrolone and stanozolol, two commonly used AAS, could have an effect on Leydig cell tumor proliferation and if their effects could be potentiated by the concomitant use of IGF‐I. Using a rat Leydig tumor cell line, R2C cells, as experimental model we found that nandrolone and stanozolol caused a dose‐dependent induction of aromatase expression and estradiol (E2) production. When used in combination with IGF‐I they were more effective than single molecules in inducing aromatase expression. AAS exhibited estrogenic activity and induced rapid estrogen receptor (ER)‐dependent pathways involving IGF1R, AKT, and ERK1/2 phosphorylation. Inhibitors for these kinases decreased AAS‐dependent aromatase expression. Up‐regulated aromatase levels and related E2 production increased cell proliferation as a consequence of increased cyclin E expression. The observation that ER antagonist ICI182,780 was also able to significantly reduce ASS‐ and AAS + IGF‐induced cell proliferation, confirmed a role for estrogens in AAS‐dependent proliferative effects. Taken together these data clearly indicate that the use of high doses of AAS, as it occurs in doping practice, enhances Leydig cell proliferation, increasing the risk of tumor development. This risk is higher when AAS are used in association with IGF‐I. To our knowledge this is the first report directly associating AAS and testicular cancer. J. Cell. Physiol. 227: 2079–2088, 2012. © 2011 Wiley Periodicals, Inc.     

Elastic fiber assembly in the adult mouse pubic symphysis during pregnancy and postpartum

Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.     

Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation

The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma membrane disorders (mean ± SEM 9.48 ± 0.65 and 1.62 ± 0.23% in raw and frozen-thawed sperm, respectively). However, both membrane peroxidation and protein phosphorylation were increased (P < 0.05) compared to raw semen (1.74 and 5.20-fold, respectively). There was a correlation (r = 0.73; P < 0.05) between the increase in lipid peroxidation and tyrosine phosphorylation. Seminal plasma, regardless of origin, reduced (P > 0.05) tyrosine phosphorylation present on the surface of cryopreserved sperm; however, lipid peroxidation was not significantly reduced. In conclusion, we inferred that emergence of phosphorylated proteins on the surface of cryopreserved sperm was due to increased lipid peroxidation that occurred during the freezing/thawing process; however, reduced tyrosine phosphorylation that occurred after addition of seminal plasma was triggered by other mechanisms, apparently independent from the reduction in membrane peroxidation.