Ultrastructural features of the myotendinous junction of the sternomastoid muscle in Wistar rats: from newborn to aging

The myotendinous junction (MTJ) is a major area for transmitting force from the skeletal muscle system and acts in joint position and stabilization. This study aimed to use transmission electron microscopy to describe the ultrastructural features of the MTJ of the sternomastoid muscle in Wistar rats from newborn to formation during adulthood and possible changes with aging. Ultrastructural features of the MTJ from the newborn group revealed pattern during development with interactions between muscle cells and extracellular matrix elements with thin folds in the sarcolemma and high cellular activity evidenced through numerous oval mitochondria groupings. The adult group had classical morphological features of the MTJ, with folds in the sarcolemma forming long projections called finger-like processes and sarcoplasmic invaginations. Sarcomeres were aligned in series, showing mitochondria near the Z line in groupings between collagen fiber bundles. The old group had altered finger-like processes, thickened in both levels of sarcoplasmic invaginations and in central connections with the lateral junctions. We conclude that the MTJ undergoes intense activity from newborn to its formation during adulthood. With increasing age, changes to the MTJ were observed in the shapes of the invaginations and finger-like processes due to hypoactivity, potentially compromising force transmission and joint stability. Microsc. Res. Tech. 75:12921296, 2012. (C) 2012 Wiley Periodicals, Inc.

Headgroup specificity for the interaction of the antimicrobial peptide tritrpticin with phospholipid Langmuir monolayers

We examined the interaction of the cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) with Langmuir monolayers of zwitterionic (dipalmitoyl phosphatidylcholine, DPPC, and dipalmitoyl phosphatidylethanolamine, DPPE) and negatively charged phospholipids (dipalmitoyl phosphatidic acid, DPPA, and dipalmitoyl phosphatidylglycerol, DPPG). Both surface pressure and surface potential isotherms became more expanded upon addition of TRP3 (DPPE similar to DPPC << DPPA < DPPG). The stronger interaction with negatively charged phospholipids agrees with data for vesicles and planar lipid bilayers, and with AMPs greater activity against bacterial membranes versus mammalian cell membranes. Considerable expansion of negatively charged monolayers occurred at 10 and 30 mol% TRP3, especially at low surface pressure. Moreover, a difference was observed between PA and PG, demonstrating that the interaction, besides being modulated by electrostatic interactions, displays specificity with regard to headgroup, being more pronounced in the case of PG, present in large quantities in bacterial membranes. In previous studies, it was proposed that the peptide acts by a toroidal pore-like mechanism [1,2]. Considering the evidence from the literature that PG shows a propensity to form a positive curvature as do toroidal pores, the observation of TRP3's preference for the PG headgroup and the dramatic increase in area promoted by this interaction represent further support for the toroidal pore mechanism of action proposed for TRP3. (C) 2012 Elsevier B.V. All rights reserved.

Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether. LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action. (C) 2012 Elsevier Inc. All rights reserved.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

EFFECT OF HEAD GROUP AND CURVATURE ON BINDING OF THE ANTIMICROBIAL PEPTIDE TRITRPTICIN TO LIPID MEMBRANES

In this work we examine the interaction between the 13-residue cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) and model membranes of variable lipid composition. The effect on peptide conformational properties was investigated by means of CD (circular dichroism) and fluorescence spectroscopies. Based on the hypothesis that the antibiotic acts through a mechanism involving toroidal pore formation, and taking into account that models of toroidal pores imply the formation of positive curvature, we used large unilamellar vesicles (LUV) to mimic the initial step of peptide-lipid interaction, when the peptide binds to the bilayer membrane, and micelles to mimic the topology of the pore itself, since these aggregates display positive curvature. In order to more faithfully assess the role of curvature, micelles were prepared with lysophospholipids containing (qualitatively and quantitatively) head groups identical to those of bilayer phospholipids. CD and fluorescence spectra showed that, while TRP3 binds to bilayers only when they carry negatively charged phospholipids. binding to micelles occurs irrespective of surface charge, indicating that electrostatic interactions play a less predominant role in the latter case. Moreover, the conformations acquired by the peptide were independent of lipid composition in both bilayers and micelles. However, the conformations were different in bilayers and in micelles, suggesting that curvature has an influence on the secondary structure acquired by the peptide. Fluorescence data pointed to an interfacial location of TRP3 in both types of aggregates. Nevertheless, experiments with a water soluble fluorescence quencher suggested that the tryptophan residues are more accessible to the quencher in micelles than in bilayers. Thus, we propose that bilayers and micelles can be used as models for the two steps of toroidal pore formation. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

Background We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D 10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N ¼ 42) or not (small follicle-small CL group; SF-SCL; N ¼ 41) on D 10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 0.33 mm vs. 10.76 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 0.28 pg/mL vs. 1.27 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 0.25 ng/mL vs. 2.62 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid deltaisomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.