Identification of regions within the third FnIII-like domain of the IL-5R alpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5R alpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5R alpha (hIL-5R alpha) and mouse IL-5R alpha (mIL-5R alpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5R alpha but not the mIL-5R alpha. By using chimeras of the mIL-5R alpha and hIL-5R alpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5R alpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5R alpha to mIL-5R alpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity, These results show that regions of the third FnIII domain of IL-5R alpha are involved in binding, in addition to the regions in domains one and two of the IL-5R alpha that were identified in an earlier study. (C) 2000 Academic Press.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in

Recognition of cyclic, acyclic, exocyclic, and spiro Acetals via structurally diagnostic ion/molecule reactions with the (CH3)(2)N-C+=O acylium ion

Reactions of the model acylium ion (CH3)(2)N-C+=O with acyclic, exocyclic, and Spiro acetals of the general formula (RO)-O-1-(CRR4)-R-3-OR2-upole mass spectrometry. Characteristic intrinsic reactivities were observed for each of these classes of acetals. The two most Characteristic intrinsic reactivities were observed for each of these classes of acetals. The two most common reactions observed were hydride and alkoxy anion [(RO-)-O-1 and (RO-)-O-2] abstraction. Other specific reactions were also observed: (a) a secondary polar [4(+) + 2] cycloaddition for acetals bearing alpha,beta-unsaturated R-3 or R-4 substituents and (b) OH- abstraction for exocyclic and spiro acetals. These structurally diagnostic reactions, in conjunction with others observed previously for cyclic acetals, are shown to reveal the class of the acetal molecule and its ring type and substituents and to permit their recognition and distinction from other classes of isomeric molecules.

Unraveling the Na,K-ATPase alpha(4) Subunit Assembling Induced by Large Amounts of C(12)E(8) by Means of Small-Angle X-ray Scattering

In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alpha beta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase AN INTEGRATED, ANIMATED MODEL

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na(+) and K(+) translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P(2c)-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E(1)/E(2)-ATPase as it undergoes conformational changes between the E(1) and E(2) forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger`s scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na(+) and K(+) translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the ""core engine"" of the pump, with respect to ATP binding, cation transport, and ADP and P(i) release.

HUMAN CHOLESTERYL ESTER TRANSFER PROTEIN EXPRESSION ENHANCES THE MOUSE SURVIVAL RATE IN AN EXPERIMENTAL SYSTEMIC INFLAMMATION MODEL: A NOVEL ROLE FOR CETP

Mice expressing human cholesteryl ester transfer protein (huCETP) are more resistant to Escherichia coli bacterial wall LIPS because death rates 5 days after intraperitoneal inoculation of LIPS were higher in wild-type than in huCETP(+/-) mice, whereas all huCETP(+/+) mice remained alive. After LIPS inoculation, plasma concentrations of TNF-alpha and IL-6 increased less in huCETP(+/+) than in wild-type mice. LPS in vitro elicited lower TNF-alpha production by CETP expressing than by wild-type macrophages. In addition, TNF-alpha production by RAW 264.7 murine macrophages increased on incubation with LPS but decreased in a dose-dependent manner when human CETP was added to the medium. Human CETP in vitro enhanced the LIPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused C-14-LPS from Salmonella typhimurium was greater in huCETP(+/+) than in wild-type mice. Present data indicate for the first time that CETP is an endogenous component involved in the first line of defense against an exacerbated production of proinflammatory mediators.

Human breast tumor slices: A model for identification of vitamin D regulated genes in the tumor microenvironment

While many studies have addressed the direct effects of 1 alpha,25(OH)(2)D(3) on breast cancer (BC) cells, stromal-epithelial interactions, which are important for the tumor development, have been largely ignored. In addition, high concentrations of the hormone, which cannot be attained in vivo, have been used. Our aim was to establish a more physiological breast cancer model, represented by BC tissue slices, which maintain epithelial-mesenchymal interactions, cultured with a relatively low 1 alpha,25(OH)(2)D(3) concentration, in order to evaluate the vitamin D pathway. Freshly excised human BC samples were sliced and cultured in complete culture media containing vehicle, 0.5 nM or 100 nM 1 alpha,25(OH)(2)D(3) for 24 h. BC slices remained viable for at least 24 h, as evaluated by preserved tissue morphology in hematoxylin and eosin (HE) stained sections and bromodeoxyuridine (BrdU) incorporation by 10% of tumor cells. VDR mRNA expression was detected in all samples and CYP24A1 mRNA expression was induced by 1 alpha,25(OH)(2)D(3) in both concentrations (but mainly with 100 nM). Our results indicate that the vitamin D signaling pathway is functional in BC slices, a model which preserves stromal-epithelial interactions and mimics in vivo conditions. (C) 2010 Elsevier Ltd. All rights reserved.

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]

Inducible nitric oxide synthase inhibition attenuates lung tissue responsiveness and remodeling in a model of chronic pulmonary inflammation in guinea pigs

We evaluated the influence of iNOS-derived NO on the mechanics, inflammatory, and remodeling process in peripheral lung parenchyma of guinea pigs with chronic pulmonary allergic inflammation. Animals treated or not with 1400W were submitted to seven exposures of ovalbumin in increasing doses. Seventy-two hours after the 7th inhalation, lung strips were suspended in a Krebs organ bath, and tissue resistance and elastance measured at baseline and after ovalbumin challenge. The strips were submitted to histopathological measurements. The ovalbumin-exposed animals showed increased maximal responses of resistance and elastance (p < 0.05), eosinophils counting (p < 0.001), iNOS-positive cells (p < 0.001), collagen and elastic fiber deposition (p < 0.05), actin density (p < 0.05) and 8-iso-PGF2 alpha expression (p < 0.001) in alveolar septa compared to saline-exposed ones. Ovalbumin-exposed animals treated with 1400 W had a significant reduction in lung functional and histopathological findings (p < 0.05). We showed that iNOS-specific inhibition attenuates lung parenchyma constriction, inflammation, and remodeling, suggesting NO-participation in the modulation of the oxidative stress pathway. (C) 2008 Elsevier B.V. All rights reserved.

Different strains of mice present distinct lung tissue mechanics and extracellular matrix composition in a model of chronic allergic asthma

The impact of genetic factors on asthma is well recognized but poorly understood. We tested the hypothesis that different mouse strains present different lung tissue strip mechanics in a model of chronic allergic asthma and that these mechanical differences may be potentially related to changes of extracellular matrix composition and/or contractile elements in lung parenchyma. Oscillatory mechanics were analysed before and after acetylcholine (ACh) in C57BL/10, BALB/c, and A/J mice, subjected or not to ovalbumin sensitization and challenge. In controls, tissue elastance (E) and resistance (R), collagen and elastic fibres` content, and alpha-actin were higher in A/J compared to BALB/c mice, which, in turn, were more elevated than in C57BL/10. A similar response pattern was observed in ovalbumin-challenged animals irrespective of mouse strain. E and R augmented more in ovalbumin-challenged A/J [E: 22%, R: 18%] than C57BL/10 mice [E: 9.4%, R: 11 %] after ACh In conclusion, lung parenchyma remodelled differently yielding distinct in vitro mechanics according to mouse strain. (C) 2008 Elsevier B.V. All rights reserved.

Assisted ventilation modes reduce the expression of lung inflammatory and fibrogenic mediators in a model of mild acute lung injury

The goal of the study was to compare the effects of different assisted ventilation modes with pressure controlled ventilation (PCV) on lung histology, arterial blood gases, inflammatory and fibrogenic mediators in experimental acute lung injury (ALI). Paraquat-induced ALI rats were studied. At 24 h, animals were anaesthetised and further randomized as follows (n = 6/group): (1) pressure controlled ventilation mode (PCV) with tidal volume (V (T)) = 6 ml/kg and inspiratory to expiratory ratio (I:E) = 1:2; (2) three assisted ventilation modes: (a) assist-pressure controlled ventilation (APCV1:2) with I:E = 1:2, (b) APCV1:1 with I:E = 1:1; and (c) biphasic positive airway pressure and pressure support ventilation (BiVent + PSV), and (3) spontaneous breathing without PEEP in air. PCV, APCV1:1, and APCV1:2 were set with P (insp) = 10 cmH(2)O and PEEP = 5 cmH(2)O. BiVent + PSV was set with two levels of CPAP [inspiratory pressure (P (High) = 10 cmH(2)O) and positive end-expiratory pressure (P (Low) = 5 cmH(2)O)] and inspiratory/expiratory times: T (High) = 0.3 s and T (Low) = 0.3 s. PSV was set as follows: 2 cmH(2)O above P (High) and 7 cmH(2)O above P (Low). All rats were mechanically ventilated in air and PEEP = 5 cmH(2)O for 1 h. Assisted ventilation modes led to better functional improvement and less lung injury compared to PCV. APCV1:1 and BiVent + PSV presented similar oxygenation levels, which were higher than in APCV1:2. Bivent + PSV led to less alveolar epithelium injury and lower expression of tumour necrosis factor-alpha, interleukin-6, and type III procollagen. In this experimental ALI model, assisted ventilation modes presented greater beneficial effects on respiratory function and a reduction in lung injury compared to PCV. Among assisted ventilation modes, Bi-Vent + PSV demonstrated better functional results with less lung damage and expression of inflammatory mediators.

Histopathology, humoral and cellular immune response in the murine model of Leishmania (Viannia) shawi

Leishmania (Viannia) shawl was recently characterized and few studies concerning modifications in cellular and humoral immune responses in experimental leishmaniasis have been conducted. In this work, immunopathological changes induced by L. shawl in chronically infected BALB/c mice were investigated. Infected BALB/c mice developed increased lesion size associated with strong inflammatory infiltrate diffusely distributed in the dermis, with highly infected macrophages. The humoral immune response was predominantly directed toward the IgG1 isotype. The functional activity of CD4(+) and CD8(+) T cells showed significantly increased TNF-alpha mRNA levels associated with reduced IFN-gamma expression by CD4(+) T cells and the double negative (dn) CD4CD8 cell subset. High IL-4 levels expressed by CD8(+) T cells and dnCD4CD8 and TGF-beta by CD4(+) and CD8(+) T cells were detected, while IL-10 was highly expressed by all three cell subpopulations. Taken together, these results show an evident imbalance between TNF-alpha and IFN-gamma that is unfavorable to amastigote replication control. Furthermore, L. shawi seems to regulate different cell populations to express deactivating cytokines to avoid its own destruction. This study indicates BALB/c mice as a potentially good experimental model for further studies on American cutaneous leishmaniosis caused by L. shawi. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Role of cytokines in mediating mechanical hypernociception in a model of delayed-type hypersensitivity in mice

In the present study, we used the electronic version of the von Frey test to investigate the role of cytokines (TNF-alpha and IL-1 beta) and chemokines (KC/CXCL-1) in the genesis of mechanical hypernociception during antigen-induced inflammation in mice. The nociceptive test consisted of evoking a hindpaw flexion reflex with a hand-held force transducer (electronic anesthesiometer) adapted with a 0.5 mm(2) polypropylene tip. The intraplantar administration of methylated bovine serum albumin (mBSA) in previously immunized (IM), but not in sham-immunized (SI) mice, induced mechanical hypernociception in a dose-dependant manner. Hypernociception induced by antigen was reduced in animals pretreated with IL-lra and reparixin (a non-competitive allosteric inhibitor of CXCR2), and in TNF receptor type 1 deficient (TNFR1-/-) mice. Consistently, antigen challenge induced a time-dependent release of TNF-alpha, IL-1 beta and KC/CXCL-1 in IM, but not in SI, mice. Consistently, antigen challenge induced a time-dependent release of TNF-alpha, IL-1 beta and KC/CXCL-1 in IM, but not in SI, mice. The increase in TNF-alpha levels preceded the increase in IL-1 beta and KC/CXCL1. Antigen-induced release of IL-1 beta and KC/CXCL1 was reduced in TNFR1-/- mice, and TNF-alpha induced hypernociception was inhibited by IL-lra and reparixin. Hypernociception induced by IL-1 beta in immunized mice was inhibited by indomethacin, whereas KC/CXCL1-induced hypernociception was inhibited by indomethacin and guanethidine, Antigen-induced hypernociception was reduced by indomethacin and guanethidine and abolished by the two drugs combined. Together, these results suggest that inflammation associated with an adaptive immune response induces hypernociception that is mediated by an initial release of TNF-alpha, which triggers that subsequent release of IL-1 beta and KC/CXCL1. The latter cytokines in turn stimulate the release of the direct-acting final mediator, prostanoids and sympathetic amines. (C) 2008 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

Role of cytokines (TNF-alpha, IL-1 beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide

Introduction Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Materials and methods Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and KC ELISA. Results CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1 beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1 beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. Conclusion These results suggest an important role of TNF-alpha, IL-1 beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.