Short communication: Red deer (Cervus elaphus) colostrum during its transition to milk.

We studied changes in chemical composition, somatic cell count, and immunoglobulin G (IgG) and M (IgM) content in red deer (Cervus elaphus) colostrum during the transition to milk at different times after parturition (<5 h, 24 h, 48 h, 2 wk, and 4 wk). The production level was higher at 2 and 4 wk of lactation than during the first day after parturition, with intermediate values at 48 h postpartum. Fat content did not vary during the study period. However, total protein and casein contents were particularly high in the initial 5 h after parturition, decreasing to approximately 50% after 24 h postpartum. Conversely, lactose concentration was low in the beginning (<5 h), increasing gradually throughout the study. Similarly, dry matter dropped during the first 24 h and then remained constant throughout the study. Urea content decreased during the study, showing a slight recovery at 4 wk. Somatic cell count was higher during the first hours after parturition and gradually decreased throughout the study period. The IgG content was higher before 5 h postpartum than at 24 h postpartum. After 5 h, the level of IgG decreased progressively until it reached 0.18 mg/mL at 4 wk of lactation. We observed a similar pattern for IgM content, but it decreased more quickly than IgG and was not detected after 2 wk. In the case of deer, milk should be considered transitional from 24 to 48 h after parturition, and samples collected after 2 wk can be considered mature milk.

PEGylated Nanoemulsions for Oral Delivery: Role of the Inner Core on the Final Fate of the Formulation.

Since the past decade, there has been growing interest to grant nanoparticles with diffusion properties across mucosae. In this sense, the nonionic block copolymer Pluronic F127 (PF127) has emerged as a promising coating agent to formulate mucus-penetrating particles. In the journey to find efficient coating agents, researchers have focused more on the effect of the coating agent architecture rather than on the role of the physicochemical properties of the nanoparticle used as the substrate. The current knowledge about mucodiffusive particles is in general based on model-like nanoparticles, such as polystyrene or poly(lactic-co-glycolic) acid nanoparticles, but there is a lack of information about the potential of PF127 on other colloidal systems. This work aims to shed some light on this issue by selecting three oils, palm (solid), coconut (semisolid), and wheat germ (liquid), with different physicochemical properties to formulate PF127-coated nanoemulsions. The obtained nanoemulsions were characterized, and their colloidal stability was tested. Their diffusion capacity was determined by particle tracking after challenging the nanoemulsions across an intestinal porcine mucus layer. In accordance with the evidence of model-like nanoparticles, our results state that PF127 allows mucodiffusion, but its effectiveness as a coating agent clearly depends on the physicochemical properties of the nanostructure core over which PF127 is placed. Among other physicochemical properties, the results certainly showed that the hydrophobic character of the nanostructure core emerges as a critical factor in the formulation of successful PF127 coatings.

[Correlation between prenatal ultrasound and postnatal diagnosis of birth defects]. / Correlación entre la ecografía prenatal y el diagnóstico postnatal de las malformaciones congénitas.

OBJECTIVES: To assess the accuracy of prenatal ultrasound diagnosis and to analyze the protocol applied for congenital defects (CD) in our environment. METHODS: Descriptive study of prenatally diagnosed CD in our area between 2004-2013. Includes: total births, fetal medicine referrals (number of consultations, ultrasound, invasive techniques) anatomical and chromosomal abnormalities, confirmed diagnoses, necropsies performed, false diagnoses, absence of prenatal diagnoses, and number and reasons for abortions (VIEs). RESULTS: Mean annual births were 3,646 ± 1,299, with a mean prenatal ultrasound of 2,144 ± 307 and 512 ± 74 invasive techniques per year. The annual average of prenatal chromosomopathies diagnosed were 26 ± 8 and 140 ± 14 anatomical abnormalities, which represents a 36.44% from all of the prenatal ultrasound performed. These include: neurological, cardiac and nephron-urological anatomic anomalies. Pre and post-natal correlation was observed in 95.6% of the DCs detected. Most common causes of abortion were chromosomal abnormalities, heart and neurological diseases. CONCLUSIONS: Due to the variety of CD that cause VIEs, a highly specialized multidisciplinary approach is recommended to ensure optimal information for parents.

OBJETIVOS: Valorar la precisión del diagnóstico ecográfico prenatal y analizar el protocolo de actuación frente a un determinado defecto congénito (DC) en nuestro medio. MATERIAL Y METODOS: Estudio descriptivo de los DC diagnosticados prenatalmente en nuestra área sanitaria entre los años 2004-2013. Como variables del estudio se incluyeron el número de nacimientos totales, derivaciones a medicina fetal (número de consultas, ecografías, técnicas invasivas) anomalías anatómicas por sistemas, cromosomopatías, diagnósticos confirmados, necropsias realizadas, falsos diagnósticos, ausencia de diagnóstico prenatal, número y motivo de interrupciones voluntarias del embarazo (IVEs). RESULTADOS: Durante el período estudiado, la media de nacimientos anuales fue de 3.646 ± 1.299, con una media de 2.144 ± 307 ecografías prenatales y 512 ± 74 técnicas invasivas anuales. La media anual de diagnóstico prenatal fue de 26 ± 8 cromosomopatías y 140 ± 14 anomalías anatómicas que suponen un 36,44% del total de las ecografías prenatales realizadas. Entre estas últimas se observaron malformaciones del sistema urinario, cardíacas y neurológicas, entre otras. Se observó correlación pre y postnatal en el 95,6% de los DC detectados. Las causas de interrupción del embarazo más frecuentes fueron las cromosomopatías, seguidas de las malformaciones congénitas (MC) cardíacas y neurológicas. CONCLUSIONES: Debido a la variedad de DC que originan IVE, es recomendable un abordaje multidisciplinar altamente especializado que garantice una información óptima a los padres.

Evidence for structural and functional abnormality in the subgenual anterior cingulate cortex in major depressive disorder.

BACKGROUND: The subgenual anterior cingulate cortex (sgACC) is considered to be an important site of abnormality in major depressive disorder. However, structural alterations in this region have not been a consistent finding and functional imaging studies have also implicated additional areas. METHOD: A total of 32 patients with major depressive disorder, currently depressed, and 64 controls underwent structural imaging with MRI. Also, 26 patients and 52 controls were examined using functional magnetic resonance imaging (fMRI) during performance of the n-back working memory task. Structural and functional changes were evaluated using whole-brain, voxel-based methods. RESULTS: The depressed patients showed volume reductions in the sgACC and orbitofrontal cortex bilaterally, plus in both temporal poles and the hippocampus/parahippocampal gyrus on the left. Functional imaging revealed task-related hypo-activation in the left lateral prefrontal cortex and other regions, as well as failure of deactivation in a subcallosal medial frontal cortical area which included the sgACC. CONCLUSIONS: Whole-brain, voxel-based analysis finds evidence of both structural and functional abnormality in the sgACC in major depressive disorder. The fact that the functional changes in this area took the form of failure of deactivation adds to previous findings of default mode network dysfunction in the disorder.

Phylogenetics and phylogeography of Euphorbia canariensis reveal an extreme Canarian-Asian disjunction but limited inter-island colonization.

Euphorbia canariensis is an iconic endemic species representative of the lowland xerophytic communities of the Canary Islands. It is widely distributed in the archipelago despite having diasporas unspecialized for long-distance dispersal. Here, we reconstructed the evolutionary history of E. canariensis at two levels: a time-calibrated phylogenetic analysis aimed at clarifying interspecific relationships and large-scale biogeographic patterns; and a phylogeographic study focused on the history of colonization across the Canary Islands. For the phylogenetic study, we sequenced the ITS region for E. canariensis and related species of Euphorbia sect. Euphorbia. For the phylogeographic study, we sequenced two cpDNA regions for 28 populations representing the distribution range of E. canariensis. The number of inter-island colonization events was explored using PAICE, a recently developed method that includes a sample size correction. Additionally, we used species distribution modelling (SDM) to evaluate environmental suitability for E. canariensis through time. Phylogenetic results supported a close relationship between E. canariensis and certain Southeast Asian species (E. epiphylloides, E. lacei, E. sessiliflora). In the Canaries, E. canariensis displayed a west-to-east colonization pattern, not conforming to the "progression rule", i.e. the concordance between phylogeographic patterns and island emergence times. We estimated between 20 and 50 inter-island colonization events, all of them in the Quaternary, and SDM suggested a late Quaternary increase in environmental suitability for E. canariensis. The extreme biogeographic disjunction between Macaronesia and Southeast Asia (ca. 11,000 km) parallels that found in a few other genera (Pinus, Dracaena). We hypothesize that these disjunctions are better explained by extinction across north Africa and southwest Asia rather than long-distance dispersal. The relatively low number of inter-island colonization events across the Canaries is congruent with the low dispersal capabilities of E. canariensis.

Matrix metalloproteinase-10 effectively reduces infarct size in experimental stroke by enhancing fibrinolysis via a thrombin-activatable fibrinolysis inhibitor-mediated mechanism.

BACKGROUND: The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate in thrombus dissolution and extracellular matrix proteolysis. The plasminogen/plasmin system activates MMPs, and some MMPs have been involved in the dissolution of fibrin by targeting fibrin(ogen) directly or by collaborating with plasmin. MMP-10 has been implicated in inflammatory/thrombotic processes and vascular integrity, but whether MMP-10 could have a profibrinolytic effect and represent a promising thrombolytic agent is unknown. METHODS AND RESULTS: The effect of MMP-10 on fibrinolysis was studied in vitro and in vivo, in MMP-10-null mice (Mmp10(-/-)), with the use of 2 different murine models of arterial thrombosis: laser-induced carotid injury and ischemic stroke. In vitro, we showed that MMP-10 was capable of enhancing tissue plasminogen activator-induced fibrinolysis via a thrombin-activatable fibrinolysis inhibitor inactivation-mediated mechanism. In vivo, delayed fibrinolysis observed after photochemical carotid injury in Mmp10(-/-) mice was reversed by active recombinant human MMP-10. In a thrombin-induced stroke model, the reperfusion and the infarct size in sham or tissue plasminogen activator-treated animals were severely impaired in Mmp10(-/-) mice. In this model, administration of active MMP-10 to wild-type animals significantly reduced blood reperfusion time and infarct size to the same extent as tissue plasminogen activator and was associated with shorter bleeding time and no intracranial hemorrhage. This effect was not observed in thrombin-activatable fibrinolysis inhibitor-deficient mice, suggesting thrombin-activatable fibrinolysis inhibitor inactivation as one of the mechanisms involved in the MMP-10 profibrinolytic effect. CONCLUSIONS: A novel profibrinolytic role for MMP-10 in experimental ischemic stroke is described, opening new pathways for innovative fibrinolytic strategies in arterial thrombosis.

Multicenter study on multidisciplinary committees in advanced prostate cancer.

INTRODUCTION AND OBJECTIVE: Although Multidisciplinary Teams (MDTs) are recommended in the management of Advanced Prostate Cancer (APC), their functioning in real practice has been poorly evaluated. We carried out a multicenter study with the objective of evaluating the functioning of uro-oncology MDTs in 6 hospitals. MATERIALS AND METHODS: A descriptive cross-sectional study was performed. The level of Compliance with the Fundamental Quality Requirements (CFQR) of the MDTs was evaluated by means of a questionnaire filled out by the coordinators of the MDTs in each hospital. The information on the perspective of the members of the MDTs was evaluated through an anonymous survey. RESULTS: A high level of CFQR in MDTs was evidenced (75%), showing deficiencies in terms of protocol update, agendas, audits, and scientific production. The survey was answered by 62.32% of the 69 physicians surveyed (urologists, oncologists, radiation therapists, radiologists, and pathologists). The 88.4% consider the duration of the meetings appropriate. There are disparate opinions concerning the protection of the MDT meeting time as well as protocol update. Of the patients with APC presented at the MDTs meeting, 62,8% require intervention from two specialties. Only 50% of respondents believe that all CRPC cases are discussed and that there is a prior agenda. The decisions made by the MDTs are reflected in the clinical history in 65.1% and are binding only in 60.5% of the cases. Half of the respondents have not been trained in MDTs. Most participants (90.7%) agree on the fact that MDTs. convey benefits. CONCLUSIONS: The evaluations of the MDTs identify rectifiable deficiencies by modifying hospital inertia and care planning.

Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration.

The peritoneal mesothelium exhibits a high regenerative ability. Peritoneal regeneration is concomitant with the appearance, in the coelomic cavity, of a free-floating population of cells whose origin and functions are still under discussion. We have isolated and characterized this cell population and we have studied the process of mesothelial regeneration through flow cytometry and confocal microscopy in a murine model lethally irradiated and reconstituted with GFP-expressing bone marrow cells. In unoperated control mice, most free cells positive for mesothelin, a mesothelial marker, are green fluorescent protein (GFP). However, 24 hrs after peritoneal damage, free mesothelin(+)/GFP(+) cells appear in peritoneal lavages. Cultured lavage peritoneal cells show colocalization of GFP with mesothelial (mesothelin, cytokeratin) and fibroblastic markers. Immunohistochemical staining of the peritoneal wall also revealed colocalization of GFP with mesothelial markers and with procollagen-1 and smooth muscle α-actin. This was observed in the injured area as well as in the surrounding not-injured peritoneal surfaces. These cells, which we herein call peritoneal repairing cells (PRC), are very abundant 1 week after surgery covering both the damaged peritoneal wall and the surrounding uninjured area. However, they become very scarce 1 month later, when the mesothelium has completely healed. We suggest that PRC constitute a type of monocyte-derived cells, closely related with the tissue-repairing cells known as 'fibrocytes' and specifically involved in peritoneal reparation. Thus, our results constitute a synthesis of the different scenarios hitherto proposed about peritoneal regeneration, particularly recruitment of circulating progenitor cells and adhesion of free-floating coelomic cells.

Assessment of three commercial ELISAs for the detection of antibodies against Porcine epidemic diarrhea virus at different stages of the immune response.

Three commercial ELISAs -two based on spike (E1 and E3) and one on nucleocapsid protein (E2)-were used to analyze the development and persistence of antibodies against Porcine epidemic diarrhea virus (PEDV). Seventy-five four-week-old PEDV-negative piglets were inoculated orally with a European G1b PEDV (INOC) and fourteen were kept as controls (CTRL). After the inoculation, E3 detected positive animals as soon as 7 days post inoculation (dpi), while the earliest detection with E1 and E2 was at 14 dpi. All samples were positive at 21 and 28 dpi using E1 and E3, respectively, while E2 failed to detect 23.3 % of the inoculated pigs at any time point. The percentages of positive samples were different through the study: E1 and E3 > E2 from 14 to 56 dpi; and E3 > E1 > E2 from 56 to 154 dpi (P < 0.05). Five months after the inoculation, E3 still detected 92.0 % (IC95 % = 85.1-98.8 %) of pigs as positive, while E1 and E2 detected only 27.0 % (IC95 % = 16.0-37.9 %) and 0%, respectively. The sensitivity for E2 never exceeded 0.62. Specificity was 1 for all ELISAs. These different outcomes could be related to the ELISA strategies (indirect versus competition), the antigens used, the cut-off, or to other intrinsic factors of each test. The observed differences could be of importance when assessing whether older animals, such as fatteners or gilts, had previously been in contact with PEDV.

The embryonic epicardium: an essential element of cardiac development.

The epicardium has recently been identified as an active and essential element of cardiac development. Recent reports have unveiled a variety of functions performed by the embryonic epicardium, as well as the cellular and molecular mechanisms regulating them. However, despite its developmental importance, a number of unsolved issues related to embryonic epicardial biology persist. In this review, we will summarize our current knowledge about (i) the ontogeny and evolution of the epicardium, including a discussion on the evolutionary origins of the proepicardium (the epicardial primordium), (ii) the nature of epicardial-myocardial interactions during development, known to be essential for myocardial growth and maturation, and (iii) the contribution of epicardially derived cells to the vascular and connective tissue of the heart. We will finish with a note on the relationships existing between the primordia of the viscera and their coelomic epithelial lining. We would like to suggest that at least a part of the properties of the embryonic epicardium are shared by many other coelomic cell types, such that the role of epicardium in cardiac development is a particular example of a more general mechanism for the contribution of coelomic and coelomic-derived cells to the morphogenesis of organs such as the liver, kidneys, gonads or spleen.

Variation in sclerophylly among Iberian populations of Quercus coccifera L. is associated with genetic differentiation across contrasting environments.

Evergreen oaks are an emblematic element of the Mediterranean vegetation and have a leaf phenotype that seems to have remained unchanged since the Miocene. We hypothesise that variation of the sclerophyll phenotype among Iberian populations of Quercus coccifera is partly due to an ulterior process of ecotypic differentiation. We analysed the genetic structure of nine Iberian populations using ISSR fingerprints, and their leaf phenotypes using mean and intracanopy plasticity values of eight morphological (leaf angle, area, spinescence, lobation and specific area) and biochemical traits (VAZ pool, chlorophyll and beta-carotene content). Climate and soil were also characterised at the population sites. Significant genetic and phenotypic differences were found among populations and between NE Iberia and the rest of the populations of the peninsula. Mean phenotypes showed a strong and independent correlation with both genetic and geographic distances. Northeastern plants were smaller, less plastic, with smaller, spinier and thicker leaves, a phenotype consistent with the stressful conditions that prevailed in the steppe environments of the refugia within this geographic area during glaciations. These genetic, phenotypic, geographic and environmental patterns are consistent with previously reported palaeoecological and common evidence. Such consistency leads us to conclude that there has been a Quaternary divergence within the sclerophyllous syndrome that was at least partially driven by ecological factors.

Parallel signal processing among mammalian MAPKs.

The intracellular signalling field is dominated by the mitogen-activated protein kinase (MAPK) cascade and its control, which involves the small GTPase Ras and sequential kinase activation. Until recently, ERK1 and ERK2 were the only cloned and well-characterized mammalian MAPKs; diverse ligand-stimulated, proline-directed protein phosphorylation events were attributed to these kinases. The recent discovery of two other MAPK subtypes, the JNK/SAPK subfamily and p38/RK (mammalian equivalents of HOG1 in yeast), reveals extreme complexity within the family and, most intriguingly, the existence in mammalian cells of parallel MAPK cascades that can be activated simultaneously.

Aß42 Peptide Promotes Proliferation and Gliogenesis in Human Neural Stem Cells.

Amyloid-ß 42 [Aß1-42 (Aß42)] is one of the main Aß peptide isoforms found in amyloid plaques of brains with Alzheimer's disease (AD). Although Aß42 is associated with neurotoxicity, it might mediate several normal physiological processes during embryonic brain development and in the adult brain. However, due to the controversy that exists in the field, relatively little is known about its physiological function. In the present work, we have analyzed the effects of different concentrations of monomeric Aß42 on cell death, proliferation, and cell fate specification of human neural stem cells (hNSCs), specifically the hNS1 cell line, undergoing differentiation. Our results demonstrate that at higher concentrations (1 µM), Aß42 increases apoptotic cell death and DNA damage, indicating that prolonged exposure of hNS1 cells to higher concentrations of Aß42 is neurotoxic. However, at lower concentrations, Aß42 significantly promotes cell proliferation and glial cell specification of hNS1 cells by increasing the pool of proliferating glial precursors, without affecting neuronal differentiation, in a concentration-dependent manner. At the molecular level, these effects could be mediated, at least in part, by GSK3ß, whose expression is increased by treatment with Aß42 and whose inhibition prevents the glial specification induced by Aß42. Since the cellular and molecular effects are known to appear decades before the first clinical symptoms, these types of studies are important in discovering the underlying pathophysiological processes involved in the development of AD. This knowledge could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.

Phenotypic expression, stability, and inheritance of a recessive resistance to monopartite begomoviruses associated with tomato yellow leaf curl disease in tomato.

Tomato-infecting begomoviruses comprise a complex of monopartite and bipartite virus species that cause severe yield and quality losses worldwide. Therefore, the availability of wide spectrum resistance for begomovirus control is desirable. However, limited sources of resistance are available. In this study, three tomato inbred lines with resistance to bipartite begomoviruses of Brazil were tested for resistance to monopartite begomoviruses associated with the tomato yellow leaf curl disease (TYLCD). Stable resistance to Tomato yellow leaf curl virus was observed either by inoculation with Bemisia tabaci or with Agrobacterium tumefaciens using an infectious clone. The resistance resulted in a complete absence of TYLCD symptoms and restricted virus accumulation. Further studies performed with the line '468-1-1-12' indicated that the resistance was also effective against three other virus species associated with TYLCD, indicating wide spectrum resistance of this source. Quantitative genetics analyses suggested that a major recessive locus with epistatic interactions is controlling the resistance to TYLCD in '468-1-1-12', which could facilitate introgression of this trait into elite tomato lines. The resistance was stable under field conditions with high TYLCD pressure. Mild symptoms could be observed in these conditions, and recovery from disease and from virus infection suggested an active host antiviral defense mechanism. The differential reaction of '468-1-1-12' against a number of TYLCD-associated viruses and artificial chimeras between them allowed to identify a region of the virus genome that presumably contains a virus determinant for breaking the resistance to infection observed in '468-1-1-12'.

[Tumor hip prosthesis in fibrous dysplasia of proximal femur. Case report]. / Prótesis tumoral de cadera en displasia fibrosa de fémur proximal. Informe de un caso.

BACKGROUND: fibrous dysplasia is characterized by the formation of fibrous tissue that generates immature malformed trabeculae. Their involvement may be monostotic or poliostotic. CASE REPORT: Thirty years old female with fibrous dysplasia of the left hip. At age 15 suffered hip fracture without apparent trauma, she was managed conservatively and significant shortening of the same limb was observed. At 30 years old, is pregnant, it had left hip pain, deformity and shortening is exacerbated. Simple radiography showed «shepherds crook deformity¼, she was treated with bisphosphonates. Computed axial tomography corroborates previous findings. She subsequently underwent subtrochanteric fracture fall with left hip dysplasia site, ground glass image of cystic appearance involving the femoral neck, trochanteric and subtrochanteric area. Tumor prosthesis of the left hip was implanted, four months evolution was favorable. CONCLUSION: Modular hip prosthesis system, reduces deformity, improves length and reduces pain in patients with resection of fibrous dysplasia of the hip.

ANTECEDENTES: La displasia fibrosa se caracteriza por la formación de tejido fibroso inmaduro que genera trabéculas mal formadas. Su afectación puede ser monostótica o poliostótica. CASO CLÍNICO: Femenino de 30 años con displasia fibrosa de cadera izquierda. A los 15 años sufre fractura de la misma cadera sin trauma aparente, manejada conservadoramente y con acortamiento importante de la misma extremidad. A los 30 años de edad se embaraza, presenta dolor en cadera izquierda y se exacerba la deformidad y el acortamiento. La radiografía simple muestra deformidad en «cayado de pastor¼, se manejó con bifosfonatos y la tomografía corrobora los hallazgos anteriores. Posteriormente sufrió caída con fractura subtrocantérica de cadera izquierda en sitio de displasia, imagen de vidrio despulido de apariencia quística que involucra el cuello femoral, zona trocantérica y subtrocantérica. Se colocó prótesis tumoral de cadera izquierda, a los cuatro meses mostró evolución favorable. CONCLUSIÓN: La prótesis tumoral femoral reduce la deformidad, mejora la longitud y disminuye el dolor en pacientes con resección de displasia fibrosa de cadera.

S. pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body.

BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic. RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis. CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.

p38/RK is essential for stress-induced nuclear responses: JNK/SAPKs and c-Jun/ATF-2 phosphorylation are insufficient.

The ERK, JNK/SAPK and p38/RK MAP kinase subtypes (reviewed in [1]) are differentially activated in mammalian cells by various stimuli, which elicit induction of immediate-early (IE) genes, such as c-fos and c-jun (reviewed in [1-3]), as well as phosphorylation of histone H3 [4] and HMG-14 [5]. Anisomycin and UV radiation have been suggested to induce c-fos and c-jun transcription via JNK/SAPK-mediated phosphorylation of TCF (ternary complex factor), for c-fos induction [6-8], and c-Jun and/or ATF-2 for c-jun induction [9-11] [12,13]. We report here that anisomycin and ultraviolet radiation (UV) activate MAP kinase kinase-6 (MKK6) [14,15], p38/RK [16] [17,18] and MAPKAP kinase-2 (MAPKAP K-2) [17-19]. By using the p38/RK inhibitor SB 203580 [20,21], we show that activation of p38/RK and/or its downstream effectors are essential for anisomycin- and UV-stimulated c-fos/c-jun induction and histone H3/HMG-14 phosphorylation, whereas JNK/SAPK activation and phosphorylation of c-Jun and ATF-2 are insufficient for these responses.

Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun.

Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.

Anisomycin selectively desensitizes signalling components involved in stress kinase activation and fos and jun induction.

Anisomycin, a translational inhibitor secreted by Streptomyces spp., strongly activates the stress-activated mitogen-activated protein (MAP) kinases JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) and p38/RK in mammalian cells, resulting in rapid induction of immediate-early (IE) genes in the nucleus. Here, we have characterized this response further with respect to homologous and heterologous desensitization of IE gene induction and stress kinase activation. We show that anisomycin acts exactly like a signalling agonist in eliciting highly specific and virtually complete homologous desensitization. Anisomycin desensitization of a panel of IE genes (c-fos, fosB, c-jun, junB, and junD), using epidermal growth factor (EGF), basic fibroblast growth factor, (bFGF), tumor necrosis factor alpha (TNF-alpha), anisomycin, tetradecanoyl phorbol acetate (TPA), and UV radiation as secondary stimuli, was found to be extremely specific both with respect to the secondary stimuli and at the level of individual genes. Further, we show that anisomycin-induced homologous desensitization is caused by the fact that anisomycin no longer activates the JNK/SAPK and p38/RK MAP kinase cascades in desensitized cells. In anisomycin-desensitized cells, activation of JNK/SAPKs by UV radiation and hyperosmolarity is almost completely lost, and that of the p38/RK cascade is reduced to about 50% of the normal response. However, all other stimuli produced normal or augmented activation of these two kinase cascades in anisomycin-desensitized cells. These data show that anisomycin behaves like a true signalling agonist and suggest that the anisomycin-desensitized signalling component(s) is not involved in JNK/SAPK or p38/RK activation by EGF, bFGF, TNF-alpha, or TPA but may play a significant role in UV- and hyperosmolarity-stimulated responses.