Validity of thoracic ultrasonography to rule out iatrogenic pneumothorax performed by pulmonologists without experience in this procedure. / Validez de la ecografía torácica para descartar neumotórax iatrogénico realizada por neumólogos sin experiencia en este procedimiento.

INTRODUCTION: Ultrasonography has been shown to be a useful tool for diagnosing pneumothorax in the hands of experts. After performing bronchopleural procedures, the recommendation is to perform chest radiography to rule out complications. Our objective was to determine the validity of thoracic ultrasonography to rule out pneumothorax after invasive procedures, conducted by pulmonologists without experience in this procedure. MATERIAL AND METHODS: Our observational prospective study consecutively included patients who underwent transbronchial biopsy (TBB), evacuating thoracentesis (ECT) and/or transparietal pleural biopsies (TPB) who were indicated subsequent chest radiography to rule out complications. In all cases, the same pulmonologist who performed the technique performed an ultrasound immediately after the procedure. A diagnosis of pneumothorax was considered the presence of a lung point or the combination of the following signs: absence of pleural sliding, absence of B-lines and presence of the «barcode¼ sign. RESULTS: We included 275 procedures (149 TBBs, 36 TPBs, 90 ECTs), which resulted in 14 (5.1%) iatrogenic pneumothoraxes. Ultrasonography presented a sensitivity of 78.5%, a specificity of 85% and a positive and negative predictive value of 22% and 98.6%, respectively. Ultrasonography did not help detect the presence of 3 pneumothoraxes, one of which required chest drainage, but adequately diagnosed 2 pneumothoraxes that were not identified in the initial radiography. CONCLUSIONS: Thoracic ultrasonography performed by pulmonologists at the start of their training helps rule out pneumothorax with a negative predictive value of 98.6%, thereby avoiding unnecessary radiographic control studies in a considerable number of cases.

Validity of lung ultrasound to rule out iatrogenic pneumothorax performed by pulmonologists without experience in this procedure.

INTRODUCTION: Ultrasonography has been shown to be a useful tool for diagnosing pneumothorax in the hands of experts. After performing bronchopleural procedures, the recommendation is to perform chest radiography to rule out complications. Our objective was to determine the validity of lung ultrasound, conducted by pulmonologists without experience in this procedure, to tule out pneumothorax after invasive procedures. MATERIAL AND METHODS: Our prospective observational study consecutively included patients who underwent transbronchial lung biopsy (TBLB), therapeutic thoracentesis (TT) and/or transparietal pleural biopsies (PB) for whom subsequent chest radiography to rule out complications was indicated. In all cases, the same pulmonologist who performed the technique performed an ultrasound immediately after the procedure. A diagnosis of pneumothorax was considered in the presence of a lung point or the combination of the following signs: absence of pleural sliding, absence of B-lines and presence of the "barcode" sign. RESULTS: We included 275 procedures (149 TBLBs, 36 BPs, 90 TTs), which resulted in 14 (5.1%) iatrogenic pneumothoraxes. Ultrasonography presented a sensitivity of 78.5%, a specificity of 85% and positive and negative predictive value of 22% and 98.6%, respectively. Ultrasonography did not help detect the presence of 3 pneumothoraxes, one of which required chest drainage, but adequately diagnosed 2 pneumothoraxes that were not identified in the initial radiography. CONCLUSIONS: Lung ultrasound performed by pulmonologists at the start of their training helps rule out pneumothorax with a negative predictive value of 98.6%, thereby avoiding unnecessary radiographic control studies in a considerable number of cases.

Undernutrition during foetal and post-natal life affects testicular structure and reduces the number of Sertoli cells in the adult rat.

To test whether undernutrition during foetal to pre-pubertal life would have long lasting effects on testicular histology in adult male offspring, eleven adult Sprague-Dawley pregnant rats were divided into two groups: Control group, n = 4, fed ad libitum, during gestation and lactation (until 25 day post-partum). Underfed group pregnant females (n = 7) were kept in cages where only dams had access to food (standard rat chow, 33.5% of ad libitum intake of Control group pregnant dams). After parturition, litters were adjusted to either 14 (Underfed group) or eight (Control group) pups. Mothers were weighed weekly. At 25 day of age pups were weaned, housed at four animals per cage, fed ad libitum and weighed weekly until euthanized at 100 day of age. Testes were processed for standard histology and morphometrical evaluation. At weaning, mother weight was lower in underfed than in Control group (mean +/- SD): 214.1 +/- 26.2 g vs 361.9 +/- 33.1 g. Body weight at 100 days of age (254 +/- 26.9 g vs 342.4 +/- 10.2 g, p

Oil spill vulnerability assessment integrating physical, biological and socio-economical aspects: application to the Cantabrian coast (Bay of Biscay, Spain).

A methodology has been developed to carry out an integrated oil spill vulnerability index, V, for coastal environments. This index takes into account the main physical, biological and socio-economical characteristics by means of three intermediate indexes. Three different integration methods (worst-case, average and survey-based) along with ESI-based vulnerability scores, V(ESI), proposed for the Cantabrian coast during the Prestige oil spill, have been analyzed and compared in terms of agreement between the classifications obtained with each one for this coastal area. Results of this study indicate that the use of the worst-case index, V(R), leads to a conservative ranking, with a very poor discrimination which is not helpful in coastal oil spill risk management. Due to the homogeneity of this coastal stretch, the rest of the methods, V(I), V(M) and V(ESI), provide similar classifications. However, V(M) and V(I) give more flexibility allowing three indexes for each coastal segment and including socio-economic aspects. Finally, the V(I) procedure is proposed here as the more advisable as using this index promotes the public participation that is a key element in the implementation of Integrated Coastal Zone Management (IZCM).

GHRH proliferative action on somatotrophs is cell-type specific and dependent on Pit-1/GHF-1 expression.

To investigate the mechanisms by which the hypothalamic peptide GHRH influences cell division, we analyzed its effects on the proliferation of two different cell lines: CHO-4, an ovary-derived cell line, and GH3, a pituitary-derived cell line. We found that GHRH induces the proliferation of pituitary-derived cells but inhibits the proliferation of ovary-derived cells. We further characterized this dual effect of GHRH to find that the cytoplasmic signals induced by this hormone are similar in both cell lines. Moreover, in CHO-4 cells GHRH stimulates two well-known positive cell cycle regulators, c-myc and cyclin D1, but is unable to induce the degradation of the negative cell cycle regulator p27(Kip1). Significantly, when the Pit-1/GHF-1 gene is exogenously expressed in CHO-4 cells, the negative effect of GHRH on the proliferation of these cells is attenuated. Furthermore, when the levels of Pit-1 are downregulated by siRNA in GH3-GHRHR cells, the positive effects of GHRH on the proliferation of these cells are diminished. These findings add to our understanding of the molecules involved in the regulation of cell proliferation by GHRH, as we demonstrate for the first time that Pit-1 is not only required to drive the expression of the GHRH receptor, as previously described, but is also needed for the downstream effects that occur after its activation to modulate cell proliferation. These data suggest that the regulation of cell proliferation in response to a specific growth factor depends in certain cell populations on the presence of a tissue-specific transcription factor.

[Tracheobronchial wall thickening secondary to herpesvirus infection in a patient with Goods syndrome]. / Engrosamiento traqueobronquial producido por herpesvirus en un paciente con síndrome de Good.

We describe an oligosymptomatic patient with Good syndrome (thymoma and hypogammaglobulinemia) in who a follow-up chest computed tomography showed circumferential tracheobronchial wall thickening. Bronchoscopy demonstrated tracheobronchitis with necrotic, vesicular and blister areas. The histopathological and immunohistochemical findings were compatible with herpes simplex virus infection. The therapeutical response to oral acyclovir was satisfactory.

Regulation of peroxisome proliferator activated receptor-gamma in rat pituitary.

Peroxisome proliferator activated-receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily and, in addition to its relation with obesity and insulin sensitivity, it has recently been localized in human and mice pituitary, indicating a functional significance of PPARgamma in adenopituitary tumours. In the present study, we localized the PPARgamma mRNA and protein in different cell types of rat pituitary. Moreover, using the real-time polymerase chain reaction, we assessed the mRNA expression of PPARgamma in different physiological and pathological settings known to be associated with alterations in anterior pituitary cell proliferation and/or function. Our experiments have shown that PPARgamma mRNA levels were repressed by oestrogen through an oestrogen receptor-alpha effect. However, PPARgamma protein levels were only modified in males but not in females. On the other hand, PPARgamma mRNA expression was increased in dwarf rats in comparison with Lewis rats. Finally, nutritional, thyroid status or pregnancy did not change PPARgamma expression. Taken together, we provide new data regarding the regulation of pituitary PPARgamma mRNA by hormonal and metabolic status.

[Bronchodilator efficacy of combined salmeterol and tiotropium in patients with chronic obstructive pulmonary disease]. / Eficacia broncodilatadora de la asociación de salmeterol y tiotropio en pacientes con EPOC.

OBJECTIVE: Bronchodilators are still the most effective drugs for controlling the symptoms of chronic obstructive pulmonary disease (COPD). Tiotropium bromide, a long-acting anticholinergic drug, has recently been added to the therapeutic arsenal for the disease. To date, there have been no studies combining 2 long-acting bronchodilators. The aim of the present trial was to determine whether the combination of salmeterol and tiotropium improved lung function in COPD patients more than either of them alone. PATIENTS AND METHODS: Twenty-two patients (20 men) diagnosed with COPD, with a mean age of 64 years, were enrolled in this cross-over trial. Active smokers were excluded. Mean (SD) forced expiratory volume in 1 second (FEV1) was 43% (14%) of predicted. All patients were experienced in the use of inhalers. The following 3 therapeutic combinations were randomly assigned to be administered for a 1-week period: a) fluticasone (500 microg/12 h), salmeterol (50 microg/12 h) and placebo; b) fluticasone, tiotropium (18 microg/24 h), and placebo; and c) fluticasone, salmeterol, and tiotropium. At the end of each period, forced spirometry was performed before inhalation of the therapeutic combination (between 8:30 am and 9:30 am) and 2 hours after inhalation. Throughout the week, morning peak flow rates measured immediately before inhalation were recorded, and there was a 48-hour wash-out period between each therapeutic combination. RESULTS: All the patients completed the protocol. There were no significant differences in preinhalation or postinhalation FEV1 with salmeterol compared to tiotropium (preinhalation FEV1, 1.17 [0.55] L compared to 1.19 [0.49] L; postinhalation FEV1, 1.32 [0.65] L compared to 1.29 [0.61] L). In all cases postinhalation FEV1 was significantly higher than preinhalation FEV1. The combination of fluticasone, salmeterol, and tiotropium proved superior to the other 2 combinations with respect to both preinhalation FEV1 and postinhalation FEV1 (preinhalation FEV1, 1.32 [0.56] L, [P<.03 in both comparisons]; postinhalation FEV1, 1.49 [0.68] L [P<.001 in both comparisons]). Peak flow rate was also significantly higher with the combination of the 2 bronchodilators (345 L/min compared to 291 L/min and 311 mL, respectively [P <.04 in both cases]). There were no notable side effects. CONCLUSIONS: In terms of improvement in lung function, the combination of salmeterol and tiotropium together with fluticasone is more effective in patients with moderate-to-severe COPD than either of the 2 bronchodilators administered alone.

Growth hormone-releasing hormone stimulates mitogen-activated protein kinase.

GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, sequestration of the betagamma-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-gamma. Despite the involvement of p21ras, the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH.

Partial characterization of a novel oestrogen-induced protein in the rat adenohypophysis.

In order to detect putative markers of prolactin-secreting pituitary tumours, adult rats were subjected to long-term oestrogenization with oestradiol benzoate (OE2) on a monthly basis. At 6 months, anterior pituitaries were dissected and incubated either as tissue fragments or as dispersed cells with a [35S]methionine mix for labelling. Proteins released into the incubation medium and from tissue extracts were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Oestrogen induced the appearance in the incubation medium of a protein (OE2 band) with an M(r) of 38,000 under reducing conditions, and high specific activity. Surprisingly, such a protein was not detected in tissue extracts. The OE2 band was detectable by 7 days after the first dose of oestrogen, and remained throughout 1 year of treatment. The tumour cell line GH3 showed a similar OE2 band which was further enhanced by oestrogens. The protein was observed similarly in both female and male pituitary donors, either intact or gonadectomized, and also in rats of different strains, suggesting that its appearance was independent of the strain of rat and gonadal status. Furthermore, the OE2 band was specific for pituitary cells and not produced by other oestrogenized tissues. No alteration in the rate of generation or the electrophoretic pattern of the OE2 band was observed when pituitary cells from oestrogenized rats were metabolically labelled while being incubated with tunicamycin. Furthermore, a system for glycan detection, adsorption to Concanavalin A or incubation with endoglycosidase F also failed to show a clear amount of glycosylation of the oestrogen-induced protein. Both immunoprecipitation experiments and time-limited proteolysis with V8 protease ruled out the possibility that the OE2 band could be structurally related to either GH or prolactin. In conclusion, oestrogens induce the generation of a new monocatenary protein with an apparent M(r) of 38,000, which has at least one intramolecular disulphide loop and is not glycosylated. The OE2 band was detected only in incubation medium and never in tissue extracts.

Conformational transition of insulin induced by n-alkyltrimethylammonium bromides in aqueous solution.

A surfactant-induced conformational transition of bovine insulin has been detected by difference spectroscopy for a homologous series of n-alkytrimethylammonium bromides, chain length C10-C16 at pH 10.0, 25 degrees C. The transition was followed as a function of surfactant concentration by absorbance measurements at 275 nm and the data were analysed to obtain the Gibbs energy of the transition in water (delta Gw degree) and in a hydrophobic environment (delta Ghc degree) for saturated protein-surfactant complexes. A value of delta Gw degree of -11.8 +/- 1.8 kJ mol-1 was found independent of n-alkyl chain length, which is similar to the value found for the n-alkylsulfate-induced transition in a previous study (-14.6 +/- 3.0 kJ mol-1). The values of delta Ghc degree were in the range approximately -88 to -100 kJ mol-1 for chain lengths from C10 to C16. The values of delta Ghc degree vs. chain length for both the n-alkyltrimethylammonium bromides and the n-alkylsulfates lie on the same curve, demonstrating that delta Ghc degree is independent of the nature of the surfactant head group.

Validez de la ecografía torácica para descartar neumotórax iatrogénico realizada por neumólogos sin experiencia en este procedimiento / Validity of lung ultrasound to rule out iatrogenic pneumothorax performed by pulmonologists without experience in this procedure

Introducción La ecografía ha demostrado ser una herramienta útil para el diagnóstico del neumotórax en manos expertas. Tras los procedimientos broncopleurales se recomienda realizar una radiografía de tórax para descartar complicaciones. Nuestro objetivo ha sido determinar la validez de la ecografía torácica para descartar neumotórax tras procedimientos invasivos, realizada por neumólogos sin experiencia en este procedimiento. Material y métodos Estudio observacional prospectivo que incluyó pacientes consecutivos sometidos a biopsia transbronquial (BTB), toracocentesis evacuadora (TE) y/o biopsias pleurales transparietales (BPT) a los que se les indicó radiografía de tórax posterior para descartar complicaciones. En todos los casos el mismo neumólogo que hizo la técnica, realizó una ecografía inmediatamente después del procedimiento. Se consideró diagnóstica de neumotórax la presencia de punto pulmonar o la combinación de los signos: ausencia de deslizamiento pleural, ausencia de líneas B y presencia del signo de «código de barras». Resultados Se incluyeron 275 procedimientos (149 BTB, 36 BPT, 90 TE) entre los que se produjeron 14 (5,1%) neumotórax iatrogénicos. La ecografía presentó una sensibilidad de 78,5%, una especificidad de 85%, y un valor predictivo positivo y negativo de 22% y 98,6%, respectivamente. La ecografía no permitió detectar la presencia de tres neumotórax, precisando uno de ellos drenaje torácico y diagnosticó adecuadamente dos neumotórax que no se detectaban en la radiografía inicial. Conclusiones La ecografía torácica realizada por neumólogos que inician su curva de aprendizaje permite descartar neumotórax con un valor predictivo negativo (VPN) del 98,6%, evitando realizar en un número considerable de casos estudios radiográficos de control innecesarios (AU)

Introduction Ultrasonography has been shown to be a useful tool for diagnosing pneumothorax in the hands of experts. After performing bronchopleural procedures, the recommendation is to perform chest radiography to rule out complications. Our objective was to determine the validity of lung ultrasound, conducted by pulmonologists without experience in this procedure, to rule out pneumothorax after invasive procedures. Material and methods Our prospective observational study consecutively included patients who underwent transbronchial lung biopsy (TBLB), therapeutic thoracentesis (TT) and/or transparietal pleural biopsies (PB) for whom subsequent chest radiography to rule out complications was indicated. In all cases, the same pulmonologist who performed the technique performed an ultrasound immediately after the procedure. A diagnosis of pneumothorax was considered in the presence of a lung point or the combination of the following signs: absence of pleural sliding, absence of B-lines and presence of the “barcode” sign. Results We included 275 procedures (149 TBLBs, 36 BPs, 90 TTs), which resulted in 14 (5.1%) iatrogenic pneumothoraxes. Ultrasonography presented a sensitivity of 78.5%, a specificity of 85% and positive and negative predictive value of 22% and 98.6%, respectively. Ultrasonography did not help detect the presence of 3 pneumothoraxes, one of which required chest drainage, but adequately diagnosed 2 pneumothoraxes that were not identified in the initial radiography. Conclusions Lung ultrasound performed by pulmonologists at the start of their training helps rule out pneumothorax with a negative predictive value of 98.6%, thereby avoiding unnecessary radiographic control studies in a considerable number of cases (AU)

[Factors determining the duration of temporary disability and return to work in a health district of Galicia]. / Determinantes de la duración de la incapacidad temporal y la vuelta al trabajo en un área sanitaria de Galicia.

OBJECTIVE: To determine the factors associated with the incidence and duration of temporary work incapacity (TWI) in a health district. DESIGN: Descriptive and retrospective study. SETTING: South health district of the province of Lugo, Spain. PARTICIPANTS: A random sample of 1513 cases was selected among the total of episodes of TWI, during 3 years period. MAIN MEASURES: The main factors analyzed are, on the one hand, the socio-demographic characteristics of the patient, his or her social security (SS) scheme, diagnosis that justifies the TWD, and the prescription date; and, on the other hand, the age, sex, specialised training, time in the post and years in practice of the physician who prescribes the TWI. The comparison of the means was carried out using variance analysis and the Kruskal-Wallis test. The relative effect of each variable on the probability of returning to the work was estimated through Cox regression models. RESULTS: The mean duration of the episodes of TWI was of 74+/-103 days. The most frequent diagnoses were those of the bones-muscles and joints (BMAJ), injuries and poisonings (IAP), and respiratory diseases (RD). The probability of returning to work is reduced with the increase of the age, with agrarian and autonomous SS affiliates, with diagnoses of mental disease or diagnoses of the circulatory system, and in cases prescribed by older doctors or less time in the post. CONCLUSIONS: The mean duration of the episodes of TWD is higher than that of other Spanish studies. The most influential factors in the return to work are the age of the patient, the SS scheme and the diagnosed illness.

Body image, risk factors for eating disorders and sociocultural influences in Spanish adolescents.

The aim was to study differences between male and female adolescents as regards body dissatisfaction, some risk factors for eating disorders, and exposure to social influences that create ideal body figures among these populations. A questionnaire comprising 40 items was administered to 240 male adolescents at 12 public and private schools in Barcelona. Twenty-nine of the questions were the same as those in another study administered to a sample of 675 female adolescents attending similar schools in the same geographical area. The other 11 questions were specifically for males. The differences between boys and girls were highly significant on almost all the items. Girls' scores were significantly higher (p = 0.000) in the following areas: dieting and exercising in order to be thin; feelings of anxiety on seeing or showing the body in public; tendency to focus on the bodies of others and on the amount of food they eat; the belief that thin people are more popular. In addition, the girls were significantly more vulnerable to potentially dangerous social influences. For the most part, males sought a heavier, more muscular body. Though a minority of males also feared being overweight, one out of four ate more than normal to gain weight and two out of three exercised to develop their muscles. The same proportion reported envying the build of certain actors. In adolescence, the ideal body figures of the sexes vary widely. This divergence reflects a greater risk of eating disorders in girls, who are also far more exposed to social situations that cause body dissatisfaction and shape risk attitudes and behaviors.

Ischemia and reperfusion enhance ATF-2 and c-Jun binding to cAMP response elements and to an AP-1 binding site from the c-jun promoter.

The transcription factors controlling the complex genetic response to ischemia and their modes of regulation are poorly understood. We found that ATF-2 and c-Jun DNA binding activity is markedly enhanced in post-ischemic kidney or in LLC-PK1 renal tubular epithelial cells exposed to reversible ATP depletion. After 40 min of renal ischemia followed by reperfusion for as little as 5 min, binding of ATF-2 and c-Jun, but not ATF-3 or CREB (cAMP response element binding protein), to oligonucleotides containing either an ATF/cAMP response element (ATF/CRE) or the jun2TRE from the c-jun promoter, was significantly increased. Binding to jun2TRE and ATF/CRE oligonucleotides occurred with an identical time course. In contrast, nuclear protein binding to an oligonucleotide containing a canonical AP-1 element was not detected until 40 min of reperfusion, and although c-Jun was present in the complex, ATF-2 was not. Incubating nuclear extracts from reperfused kidney with protein phosphatase 2A markedly reduced binding to both the ATF/CRE and jun2TRE oligonucleotides, compatible with regulation by an ATF-2 kinase. An ATF-2 kinase, which phosphorylated both the transactivation and DNA binding domains of ATF-2, was activated by reversible ATP depletion. This kinase coeluted on Mono Q column chromatography with a c-Jun amino-terminal kinase and with the peak of stress-activated protein kinase, but not p38, immunoreactivity. In conclusion, DNA binding activity of ATF-2 directed at both ATF/CRE and jun2TRE motifs is modulated in response to the extreme cellular stress of ischemia and reperfusion or reversible ATP depletion. Phosphorylation-dependent activation of the DNA binding activity of ATF-2, which appears to be regulated by the stress-activated protein kinases, may play an important role in the earliest stages of the genetic response to ischemia/reperfusion by targeting ATF-2 and c-Jun to specific promoters, including the c-jun promoter and those containing ATF/CREs.

Oleic acid blocks epidermal growth factor-activated early intracellular signals without altering the ensuing mitogenic response.

In EGFR-T17 cells, which express high levels of the epidermal growth factor (EGF) receptor, addition of a saturating dose of EGF (10 nM) leads to an increase in Ins(1,4,5)P3/diacylglycerol and also to cytosolic calcium [Ca2+]i due to both intracellular redistribution and influx from extracellular medium. Pretreatment of cells with cis-unsaturated nonesterified fatty acids such as oleic acid (1 to 100 microM) inhibited EGF-stimulated Ins(1,4,5)P3 generation and Ca2+ release from intracellular stores. Furthermore, such a treatment completely suppress Ca2+ influx in a dose-dependent manner. At doses capable of suppressing such early signals, oleic acid did not alter the process of EGF-mediated internalization of the EGF/EGF-receptor complex, suggesting that [Ca2+]i rise did not mediate receptor internalization. EGF-induced cell proliferation assessed by either thymidine incorporation into DNA, direct cell counting, and microscopic observation was not altered by oleic acid, at doses able to block EGF-mediated early signals. In conclusion, suppression of Ins(1,4,5)P3 generation and [Ca2+]i rises by oleic acid did not alter EGF-receptor internalization nor EGF-induced cell mitosis. Such results suggest that [Ca2+]i rise is not instrumental for EGF-stimulated cell proliferation.

Activation of a human Ste20-like kinase by oxidant stress defines a novel stress response pathway.

Mammalian homologs of the yeast protein kinase, Sterile 20 (Ste20), can be divided into two groups based on their regulation and structure. The first group, which includes PAK1, is regulated by Rac and Cdc42Hs, and activators have been identified. In contrast, very little is known about activators, regulatory mechanisms or physiological roles of the other group, which consists of GC kinase and MST1. We have identified a human Ste20-like kinase from the GC kinase group, SOK-1 (Ste20/oxidant stress response kinase-1), which is activated by oxidant stress. The kinase is activated by autophosphorylation and is markedly inhibited by its non-catalytic C-terminal region. SOK-1 is activated 3- to 7-fold by reactive oxygen intermediates, but is not activated by growth factors, alkylating agents, cytokines or environmental stresses including heat shock and osmolar stress. Although these data place SOK-1 on a stress response pathway, SOK-1, unlike GC kinase and PAK1, does not activate either of the stress-activated MAP kinase cascades (p38 and SAPKs). SOK-1 is the first mammalian Ste20-like kinase which is activated by cellular stress, and the activation is relatively specific for oxidant stress. Since SOK-1 does not activate any of the known MAP kinase cascades, its activation defines a novel stress response pathway which is likely to include a unique stress-activated MAP kinase cascade.

Partial characterization of a putative new growth factor present in pathological human vitreous.

BACKGROUND: Several growth factors have been implicated in the development of proliferative eye diseases, and some of those are present in human vitreous (HV). The effects of HV on cellular responses which modulate proliferative cell processes were studied. This study describes the partial characterization of a vitreous factor activity which does not correspond to any of the previously reported growth factors in pathological HV. METHODS: Vitreous humour was obtained from medical vitrectomies, from patients with PDR and PVR. The biological activity of the vitreous factor was determined by its ability to increase cytosolic calcium concentration ([Ca2+]i), increase production of inositol phosphates, and induce cell proliferation in the cell line EGFR T17. In some experiments other cell lines, such as NIH 3T3, 3T3-L1, FRTL5, A431, PC12, Y79, and GH3, were also employed. Measurement of [Ca2+]i in cell suspensions was performed using the fluorescent Ca2+ indicator fura-2. The activity of the factor present in HV was compared with other growth factors by means of: (a) [Ca2+]i mobilization pattern, (b) sequential homologous and heterologous desensitization of receptors, (c) effects of phorbol esters on their action, and (d) inactivation after treatment with different proteolytic enzymes. RESULTS: The HV-induced cell proliferation and increases in [Ca2+]i concentration were characterized by a peculiar time pattern. The different approaches used ruled out its identity with PDGF, bFGF, EGF, TGF-beta, IGFs, TNF-alpha, NGF, and other compounds such as ATP, angiotensin I, and bradykinin. Vitreous factor actions are mediated by specific receptors apparently regulated by PKC. This factor is able to induce [Ca2+]i mobilization in most of the cell lines studied, indicating that its effects are not tissue specific. CONCLUSIONS: These results suggest the presence of a growth factor activity in pathological HV which may be due to the presence of an undescribed growth factor in the eye.

Activation of the Ste20-like oxidant stress response kinase-1 during the initial stages of chemical anoxia-induced necrotic cell death. Requirement for dual inputs of oxidant stress and increased cytosolic [Ca2+].

Signal transduction mechanisms activated during the early stages of necrotic cell death are poorly characterized. We have recently identified the Sterile 20 (Ste20)-like oxidant stress response kinase-1, SOK-1, which is a member of the Ste20 kinase family. We report that SOK-1 is markedly activated as early as 20 min after chemical anoxia induced by exposure of Madin-Darby canine kidney or LLC-PK1 renal tubular epithelial cells to 2-deoxyglucose (2-DG) and any one of three inhibitors of the electron transport chain, cyanide (CN), rotenone, or antimycin A. Since oxidant stress activates SOK-1, we postulated that reactive oxygen species (ROS), which are produced by the electron transport chain during chemical anoxia, might be responsible for SOK-1 activation. The time course of CN/2-DG-induced SOK-1 activation and of production of ROS, measured in cells loaded with dichlorofluorescein, were compatible with a role for ROS in SOK-1 activation. Furthermore, preincubation of LLC-PK1 cells with three unrelated scavengers of ROS, pyrrolidine dithiocarbamate, pyruvate, or nordihydroguaiaretic acid, reduced both cellular oxidant stress and activation of SOK-1 by CN/2-DG. An increase in cytosolic free [Ca2+] ([Ca2+]i) was necessary but not sufficient for CN/2-DG-induced activation of SOK-1. Preincubation of cells with BAPTA-AM prevented activation of SOK-1. Incubation of cells with thapsigargin or the calcium ionophore, A23187, had no effect on SOK-1 activity, but preincubation of cells with either of these agents markedly enhanced CN/2-DG-induced activation of SOK-1 (20-fold versus 7-fold). In summary, chemical anoxia activates SOK-1 via an oxidant stress-dependent mechanism that is both critically dependent upon and markedly amplified by an increase in [Ca2+]i. This requirement for dual inputs of oxidant stress and an increase in [Ca2+]i may prevent inappropriate activation of the kinase by milder degrees of oxidant stress, which are insufficient to generate an increase in [Ca2+]i. The activation of SOK-1 may be one of the cell's earliest responses to inducers of necrotic cell death.