Structure of the haptoglobin-haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies(1), haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction(2). Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 angstrom resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected beta-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the alpha- and beta-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the alpha beta dimer is highly overlapping with the interface between the two alpha beta dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin alpha-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

Employing perichromism for probing the properties of carboxymethyl cellulose films: an expedient, accurate method for the determination of the degree of substitution of the biopolymer derivative

The properties of films of carboxymethyl cellulose, CMC, of different degree of substitution, DS, have been examined by the use of perichromic indicators (probes). The film properties that have been determined are: empirical polarity, E-T(33); "acidity", alpha; "basicity", beta; and dipolarity/polarizability, pi*. This has been achieved by employing the following perichromic probes: 4-nitroaniline, 4-nitroanisole, 4-nitro-N,N-dimethylaniline, and 2,6-dichloro-4-(2,4,6-triphenyl-pyridinium-1-yl)phenolate, WB. The correlations between both E-T(33)- or pi* and DS were found to be linear; that between beta and DS is a second order polynomial; no obvious correlation was found between alpha and DS. The polarities of CMC films are in the range of those of butyl alcohols. As models for CMC, we have employed cellulose plus CMC of high DS; oxidized cellulose with degree of oxidation = 0.5; sodium glucuronate. The former model behaved akin to CMC, but the plots of the perichromic properties versus DS showed different slopes/intercepts. FTIR data and molecular dynamics simulations on the solvation of WB have shown that this difference can be traced to more efficient hydrogen bonding between the film of the model and the probe. This affects the intra-molecular charge-transfer energy of the latter, leading to different responses to the variation of DS. Based on the excellent linear correlation between E-T(33) and DS, for CMC from different origins, we suggest that perichromism is a simple, accurate, and expedient alternative for the determination of DS of the biopolymer derivative.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Electrophysiological Responses of the Naupactus bipes Beetle to Essential Oils from Piperaceae Species

Electrophysiological responses based on electroantennographic detection (GC-EAD) and electroantennography (EAG) analysis of Naupactus bipes beetles (Germar, 1824) (Coleoptera: Curculionidae: Brachycerinae) were used to test volatile oils of Piper gaudichaudianum, P. regnellii and P. hispidum. In the EAG experiments, female and male beetles showed significant EAG response to the three volatile oils of Piper species, with the females' responses slightly higher than the males'. The experiments with GC-EAD revealed that some terpenoids (namely, alpha-pinene, beta-pinene, myrcene, alpha-copaene and germacrene) present in the leaf essential oils of the Piper species are perceptible to female and male beetles.

Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Neural differentiation of rat aorta pericyte cells

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins beta 3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain. (C) 2011 International Society for Advancement of Cytometry

Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)

(Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)). The cotyledons of Hymenaea courbaril store large amounts of xyloglucan, a cell wall polysaccharide that is believed to serve as storage for the period of seedling establishment. During storage mobilisation, xyloglucan seems to be degraded by a continuous process that starts right after radicle protrusion and follows up to the establishment of photosynthesis. Here we show evidence that events related to the hydrolases activities and production (alpha-xylosidase, beta-galactosidase, beta-glucosidase and xyloglucan endo-beta-transglucosilase) as well as auxin, showed changes that follow the diurnal cycle. The period of higher hydrolases activities was between 6pm and 6am, which is out of phase with photosynthesis. Among the enzymes, alpha-xilosidase seems to be more important than beta-glucosidase and beta-galactosidase in the xyloglucan disassembling mechanism. Likewise, the sugars related with sucrose metabolism followed the rhythm of the hydrolases, but starch levels were shown to be practically constant. A high level of auxin was observed during the night, what is compatible with the hypothesis that this hormone would be one of the regulators of the whole process. The probable biological meaning of the existence of such a complex control mechanism during storage mobilisation is likely to be related to a remarkably high level of efficiency of carbon usage by the growing seedling of Hymenaea courbaril, allowing the establishment of very vigorous seedlings in the tropical forest.

Crotamine Pharmacology Revisited: Novel Insights Based on the Inhibition of K-V Channels

Crotamine, a 5-kDa peptide, possesses a unique biological versatility. Not only has its cell-penetrating activity become of clinical interest but, moreover, its potential selective antitumor activity is of great pharmacological importance. In the past, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltage-gated potassium (K-V) channels in an effort to explain its in vivo effects. Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 K-V channels and 4 Na-V channels), expressed in Xenopus laevis oocytes. Crotamine selectively inhibits K-V 1.1, K-V 1.2, and K-V 1.3 channels with an IC50 of similar to 300 nM, and the key amino acids responsible for this molecular interaction are suggested. Our results demonstrate for the first time that the symptoms, which are observed in the typical crotamine syndrome, may result from the inhibition of K-V channels. The ability of crotamine to inhibit the potassium current through K-V channels unravels it as the first snake peptide with the unique multifunctionality of cell-penetrating and antitumoral activity combined with K-V channel-inhibiting properties. This new property of crotamine might explain some experimental observations and opens new perspectives on pharmacological uses.

Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference

Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models. (C) 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

Interferon-gamma alters the phagocytic activity of the mouse trophoblast

Interferon-gamma (IFN-gamma) mediates diverse functions in bone marrow-derived phagocytes, including phagocytosis and microbe destruction. This cytokine has also been detected at implantation sites under both physiological and pathological conditions in many different species. At these particular sites, the outermost embryonic cell layer in close contact with the maternal tissues, the trophoblast exhibits intense phagocytic activity. To determine whether IFN-gamma affects phagocytosis of mouse-trophoblast cells, ectoplacental cone-derived trophoblast was cultured and evaluated for erythrophagocytosis. Phagocytic activity was monitored ultrastructurally and expressed as percentage of phagocytic trophoblast in total trophoblast cells. Conditioned medium from concanavalin-A-stimulated spleen cells significantly enhanced trophoblast phagocytosis. This effect was blocked by pre-incubation with an anti-IFN-gamma neutralizing antibody. Introduction of mouse recombinant IFN-gamma (mrIFN-gamma) to cultures did not increase cell death, but augmented the percentage of phagocytic cells in a dose-dependent manner. Ectoplacental cones from mice deficient for IFN-gamma receptor alpha-chain showed a significant decrease of the phagocytosis, even under mrIFN-gamma stimulation, suggesting that IFN-gamma-induced phagocytosis are receptor-mediated. Reverse transcriptase-PCR analyses confirmed the presence of mRNA for IFN-gamma receptor alpha and beta-chains in trophoblast cells and detected a significant increase in the mRNA levels of IFN-gamma receptor beta-chain, mainly, when cultured cells were exposed to IFN-gamma. Immunohistochemistry and Western blot analyses also revealed protein expression of the IFN-gamma receptor alpha-chain. These results suggest that IFN-gamma may participate in the phagocytic activation of the mouse trophoblast, albeit the exact mechanism was not hereby elucidated. Protective and/or nutritional fetal benefit may result from this physiological response. In addition, our data also shed some light on the understanding of trophoblast tolerance to inflammatory/immune cytokines during normal gestation.