Deregulation of c-Src tyrosine kinase and its downstream targets in pre-eclamptic placenta.

AIM: Pre-eclampsia is a serious pregnancy disorder characterized by the new onset of hypertension and proteinuria in the second trimester of pregnancy. The determination of a key signaling regulatory mechanism involved in placental functions is critical to understanding the pathogenesis of pre-eclampsia. The aim of this study was to examine the activity of c-Src and its downstream targets, extracellular signal-regulated kinase 1/2, p38 and Jun N-terminal kinase, as well as nuclear factor (NF)-ĸB in placental tissues collected from women with pre-eclampsia. METHODS: Ten pre-eclamptic (PE) placentas and 10 control placentas were used in this study. The Western blot method was performed to evaluate the c-Src/ mitogen activated protein kinase/NF-ĸB signaling pathway in each group. RESULTS: c-Src phosphorylation at Tyr-416, used as a measure of c-Src activity, was significantly decreased in PE placentas relative to the control. Reduced c-Src activity resulted in the suppression of extracellular signal-regulated kinase 1/2 phosphorylation and a significant reduction in the phosphorylation of p38 and Jun N-terminal kinase in PE placentas. Moreover, IĸBα phosphorylation was significantly elevated, while NF-ĸB phosphorylation was suppressed in PE placentas. CONCLUSIONS: The c-Src/MAPK/NF-ĸB signaling pathway may contribute to the pathogenesis of pre-eclampsia.

Pharmacological Inactivation of Src Family Kinases Inhibits LPS-Induced TNF-α Production in PBMC of Patients with Behçet's Disease.

Behçet's disease (BD) is a multisystemic chronic inflammatory disease characterized by relapsing oral and genital ulcers, uveitis, and skin lesions. The pathogenesis of BD is still unknown. Aberrant production of some cytokines/chemokines plays an important role in the pathogenesis of various inflammatory diseases. Revealing a key signaling regulatory mechanism involved in proinflammatory cytokines/chemokines production is critical for understanding of the pathogenesis of BD. The aim of this study was to determine the role of Src family kinases (SFKs) in production of some LPS-induced proinflammatory cytokines/chemokines in peripheral blood mononuclear cells (PBMC) of active BD patients. Chemical inhibition of SFKs activity impaired LPS-induced TNF-α production in PBMC of active BD patients, suggesting that modulating SFKs activity may be a potential target for BD treatment.

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1.

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.

Ganoderma Lucidum Protects Rat Brain Tissue Against Trauma-Induced Oxidative Stress.

Objective: Traumatic brain injury causes tissue damage, breakdown of cerebral blood flow and metabolic regulation. This study aims to investigate the protective influence of antioxidant Ganoderma lucidum (G. lucidum) polysaccharides (GLPs) on brain injury in brain-traumatized rats. Methods: Sprague-Dawley conducted a head-traumatized method on rats by dropping off 300 g weight from 1 m height. Groups were categorized as control, G. lucidum, trauma, trauma+ G. lucidum (20 mL/kg per day via gastric gavage). Brain tissues were dissected from anesthetized rats 7 days after injury. For biochemical analysis, malondialdehyde, glutathione and myeloperoxidase values were measured. Results: In histopathological examination, neuronal damage in brain cortex and changes in blood brain barrier were observed. In the analysis of immunohistochemical and western blot, p38 mitogen-activated protein kinase, vascular endothelial growth factor and cluster of differentiation 68 expression levels were shown. These analyzes demonstrated the beneficial effects of GLPs on brain injury. Conclusion: We propose that GLPs treatment after brain injury could be an alternative treatment to decraseing inflammation and edema, preventing neuronal and glial cells degeneration if given in appropriate dosage and in particular time intervals.

Effects of Potentilla fulgens as a Prophylactic Agent in Tibial Defects in Rats.

OBJECTIVE: To investigate the effects of Potentilla fulgens as a prophylactic agent on tibial defects in the rat. STUDY DESIGN: Twenty-eight male Wistar albino rats weighing 200-215 g each were divided into 3 experimental groups. The tibial bone defect group served as the control group. The experimental groups were Potentilla fulgens with tibial defect (14 days) and Potentilla fulgens with tibial defect (28 days). Extract of Potentilla fulgens was mixed with water (400 mg/kg/day) and given to groups 14 and 28 as drinking water. The histopathological and immunohistochemical characteristics of each tibial bone cavity within each group were observed. The trabecular new bone formation was evaluated by expression rate of osteonectin and osteopontin. RESULTS: In the Potentilla fulgens + tibial defect group (14 days), trabecular bone had started combining extensive new bone formation, osteocyte cells were evident, and lamellar bone was formed. Osteoblasts showed a positive reaction with osteonectin. Osteopontin expression was positively observed between fibrous structures and in the osteoblast and osteocyte cells. This can be considered indicative of new bone formation. In the Potentilla fulgens + tibial defect group (28 days), an increase in expansion in trabecular bone and myeloid tissue was observed. Osteoblastic activity and osteocyte cells began to be observed in new bone fragments. CONCLUSION: In our study we show that Potentilla fulgens extract provided a protective effect on new bone formation and aided in the development of osteocytes and secretion of matrix in osteoblasts. Additionally, we show the inductive effect of the extract on new bone formation. In particular, the expression of osteopontin and osteonectin was also supported with the Western blot technique on the development of osteoblasts and osteocytes, showing a similar trend with our results.

Immunohistochemical and histopathological changes in the teeth of rats after lead acetate application.

OBJECTIVE: To study the toxic processes of lead in teeth by investigating the histopathologic and immunohistochemical effects of lead on incisor teeth of rats. STUDY DESIGN: Thirty Wistar Albino rats weighing 180-200 g were divided into 2 groups: a control group and a lead acetate group (i.e., the experimental group). The experimental group received lead acetate at 500 ppm in their drinking water for 60 days, and the control group received distilled water. The sections of the control group and the experiment group were histopathologically compared. RESULTS: The longitudinal cross-sections of the mandibular incisor teeth were analyzed. Degeneration in the periodontal membrane and vascular dilation in pulp and inflammatory cell infiltration were observed, and vimentin expression was seen in the dental pulp and the periodontal membrane. CONCLUSION: The presence of vimentin in the dentinal tubules indicated that the odontoblast processes in these tubules were positive. In the experimental group the profile of dentinal tubules was found to be impaired and disorganized. These results indicate a loss of collagen fibers in the periodontal membrane of incisor teeth in the experimental group. In the same group the gripping function of the incisor teeth was affected due to the spacing of the intermediate filaments that are located between the alveolar bone and the periodontal ligament. The expression of vimentin on the tooth was reduced by the lead acetate treatment. Lead acetate may affect the adhesion of the tooth to the alveolar bone.

Dual role of Src kinase in governing neuronal survival.

BACKGROUND: Src-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear. OBJECTIVE: To define the effect of inhibition of Src activity and expression on neuronal survival. RESULTS: In agreement with our previous findings, we demonstrated that Src was cleaved by calpain to form a 52-kDa truncated fragment in neurons undergoing excitotoxic cell death, and expression of the recombinant truncated Src fragment induced neuronal death. The data confirm that the neurotoxic signaling pathways are intact in the neurons we used for our study. To define the functional role of neuronal SFKs, we treated these neurons with SFK inhibitors and discovered that the treatment induced cell death, suggesting that the catalytic activity of one or more of the neuronal SFKs is critical to neuronal survival. Using small hairpin RNAs that suppress Src expression, we demonstrated that Src is indispensable to neuronal survival. Additionally, we found that neuronal death induced by expression of the neurotoxic truncated Src mutant, treatment of SFK inhibitors or knock-down of Src expression caused inhibition of the neuroprotective protein kinases Erk1/2, or Akt. CONCLUSIONS: Src is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases.

A biosensor of SRC family kinase conformation by exposable tetracysteine useful for cell-based screening.

We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.

Tyrosine 416 is phosphorylated in the closed, repressed conformation of c-Src.

c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.

Immunoexpression of vascular endothelial growth factor, beta-cell lymphoma 2 and cluster of differentiation 68 in cerebellar tissue of rats treated with Ganoderma lucidum / Inmunoexpresión del factor de crecimiento endotelial vascular, linfoma de células beta 2 y grupo de diferenciación 68 en tejido cerebeloso de ratas tratadas con Ganoderma lucidum

Traumatic brain injury (TBI) can potentially lead to hemorrhages in all areas of the skull, which can damage cells and nerve connections. This study aims to investigate the protective effects of Ganoderma lucidum polysaccharides (GLPS) as a antioxidant on cerebellar cell tissues after traumatic brain injury in rats. Sprague Dawley rats were subjected to TBI with a weight-drop device using 300 g1m weight-height impact. The groups are consisted of control, trauma, and trauma+Ganoderma lucidum groups. At seven days post-brain injury, experimental rats were decapitated after intraperitoneal administration of ketamine HCL (0.15 ml/100 g body weight). Cereballar samples were taken for histological examination or determination of malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) activity. Significant improvement was observed in cells and vascular structures of Ganoderma lucidum treated groups when compared to untreated groups. It is believed that Ganoderma lucidum may have an effect on the progression of traumatic brain injury. Ganoderma lucidum application may affect angiogenetic development in blood vessel endothelial cells, decrease inflammatory cell accumulation by affecting cytokine mechanism and may create apoptotic nerve cells and neuroprotective mechanism in glial cells.

La lesión cerebral traumática (LCT) puede provocar hemorragias en todas las áreas del cráneo, lo que puede dañar las células y las conexiones nerviosas. Este estudio tuvo como objetivo investigar los efectos protectores de los polisacáridos de Ganoderma lucidum (GLPS) como antioxidante en los tejidos de las células del cerebelo después de la lesión cerebral traumática en ratas. Ratas Sprague Dawley fueron sometidas a TBI con un dispositivo de caída de peso usando un impacto de peso de 300 g-1 m. Se formaron los siguientes grupos: control, trauma y trauma + Ganoderma lucidum. Siete días después de la lesión cerebral, las ratas experimentales fueron decapitadas después de la administración intraperitoneal de ketamina HCL (0,15 ml / 100 g de peso corporal). Se tomaron muestras cerebrales para el examen histológico y para la determinación de niveles de malondialdehído (MDA) y glutatión (GSH) y actividad de mieloperoxidasa (MPO). Se observó una mejora significativa en las células y las estructuras vasculares de los grupos tratados con Ganoderma lucidum en comparación con los grupos no tratados. Durante el estudio se observó que Ganoderma lucidum puede tener un efecto sobre la progresión de la lesión cerebral traumática. La aplicación de Ganoderma lucidum puede afectar el desarrollo angiogénico en las células endoteliales de los vasos sanguíneos, disminuir la acumulación de células inflamatorias al afectar el mecanismo de las citocinas y puede crear células nerviosas apoptóticas y un mecanismo neuroprotector en las células gliales.

ReAsH/FlAsH labeling and image analysis of tetracysteine sensor proteins in cells.

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).