Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta

Relaxin is a polypeptide hormone that has diverse effects on reproductive and non-reproductive tissues. Relaxin activates the G-protein coupled receptors, LGR7 and LRG8. Early studies described increased cAMP and protein kinase A activity upon relaxin treatment, but cAMP accumulation alone could not account for all of the relaxin-mediated effects. We utilized the human monocyte cell line THP-1 to study the mechanism of relaxin-stimulated CAMP production. ^ Relaxin treatment in THP-1 cells produces a biphasic time course in cAMP accumulation, where the first peak appears as early as 1–2 minutes with a second peak at 10–20 minutes. Selective inhibitors for phosphoinositide 3-kinase (P13K), such as wortmannin and LY294002, show a dose-dependent inhibition of relaxin-stimulated cAMP accumulation, specific for the second peak of the relaxin time course. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 is mediated by the activity of phosphodiesterases. Furthermore, LY294002 blocks upregulation of vascular endothelial growth factor transcript levels by relaxin. ^ To further delineate relaxin signaling pathways, we searched for downstream targets of PI3K that could activate adenylyl cyclase (AC). Protein kinase C ζ (PKCζ) was a prime candidate because it activates types II and V AC. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin-induced cAMP production to the same degree as LY294002 (∼40%). Relaxin stimulates PKCζ translocation to the plasma membrane in THP-1, MCF-7, PHM1-31, and MMC cells, as shown by immunocytochemistry. PKCζ translocation is P13K-dependent and independent of cAMP production. Antisense PKCζ oligodeoxynucleotides (PKCζ-ODNs) deplete both PKCζ transcript and protein levels in THP-1 cells. PKCζ-ODNs abolish relaxin-mediated PKCζ translocation and inhibit relaxin stimulation of cAMP by 40%, as compared to mock and random ODN controls. Treatment with LY294002 in the presence of PKCζ-ODNs results in little further inhibition. Taken together, we present a novel role for PI3K and PKCζ in relaxin stimulation of cAMP and provide the first example of the PKCζ regulation of AC in an endogenous system. Furthermore, we have identified higher order complexes of AC isoforms and PKA anchoring proteins in attempts to explain the differential coupling of relaxin to cAMP and PI3K-signaling pathways in various cell types. ^

Enzyme activity and liver tissue PCB concentrations of chicks of northern fulmars (F. glacialis) and black-legged kittiwakes (R. tridactyla) from Kongsfjorden

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO2-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher XOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold_

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity–dormancy transition.

Potenciación de la respuesta productiva en rumiantes mediante el empleo de proteínas protegidas

El principal objetivo de esta tesis fue incrementar la eficiencia proteica en las dietas de rumiantes mediante el uso de proteínas protegidas (harina de girasol y guisante de primavera), así como mejorar la predicción de los aportes de proteína microbiana. Una partida de harinas comerciales de girasol (HG) y de guisante de primavera (GP) fueron tratadas con soluciones 4 N de ácido málico (268,2 g/L) o ácido ortofosfórico (130,6 g/L). Para cada harina, ácido y día de tratamiento, dos fracciones de 12,5 kg fueron pulverizadas sucesivamente en una hormigonera con la solución de ácido correspondiente mediante un pulverizador de campo. Las dos fracciones fueron mezcladas posteriormente y se dejaron reposar durante 1 h a temperatura ambiente. La mezcla fue luego secada en una estufa de aire forzado a 120 ºC durante 1 h. La estufa fue apagada inmediatamente después y el material tratado se mantuvo dentro de ésta hasta la mañana siguiente. El material fue removido durante el proceso de secado cada 30 min durante las primeras 2 h y cada 60 min durante las 5 h posteriores. Este proceso se repitió hasta conseguir las cantidades de harinas tratadas necesarias en los distintos ensayos. En el primer experimento (capitulo 3) se llevaron a cabo estudios de digestión ruminal e intestinal para evaluar los efectos de la aplicación de las soluciones ácidas indicadas y calor a fin de proteger las proteínas de HG y GP contra la degradación ruminal. Estos estudios se realizaron con tres corderos canulados en el rumen y en el duodeno. El estudio de digestión ruminal fue realizado en tres periodos experimentales en los que los corderos fueron alimentados sucesivamente con tres dietas isoproteicas que incluían HG y GP, sin tratar o tratadas con ácidos málico u ortofosfórico. Cada periodo experimental de 21 días incluyó sucesivamente: 10 días de adaptación a las dietas, un estudio del tránsito ruminal de las partículas de HG y GP (días 11 a 14), y la incubación de las muestras de ambos alimentos en bolsas de nailon (días 15–21). Las harinas incubadas en cada periodo experimental correspondieron a las que fueron incluidas en las dietas. Las bacterias ruminales fueron marcadas desde el día 11 hasta el día 21 del periodo experimental mediante infusión intra-ruminal continua con una fuente de 15N. Tras finalizar las incubaciones in situ el día 21 el rumen fue vaciado en cada periodo para aislar las bacterias asociadas a la fase sólida y liquida del rumen. El estudio de digestión intestinal fue realizado veinte días después del final del estudio ruminal a fin de eliminar el enriquecimiento en 15N de la digesta. En este estudio se incubaron muestras compuestas obtenidas mediante la combinación de los diferentes residuos no degradados en el rumen de forma que fuesen representativas de la composición química de la fracción no degradada en el rumen (RU). En esta fase los corderos fueron alimentados con la dieta sin tratar para determinar la digestibilidad de las harinas tanto tratadas como sin tratar mediante la técnica de las bolsas móviles. Además, las proteínas contenidas en las harinas tratadas y sin tratar, así como en las muestras correspondientes a los residuos a 0 h, las muestras compuestas anteriormente indicadas y las muestras no digeridas intestinalmente fueron extraídas y sometidas a electroforesis para determinar el sitio de digestión de las diferentes fracciones proteicas. Las estimaciones de la RU y la digestibilidad intestinal de la materia seca, la materia orgánica (solamente para RU), la proteína bruta (PB) y el almidón (solamente en GP) fueron obtenidos considerando la contaminación microbiana y las tasas de conminución y salida de partículas. Las estimaciones de RU y de la digestibilidad intestinal disminuyeron en todas las fracciones evaluadas de ambos alimentos al corregir por la contaminación microbiana acaecida en el rumen. Todas las estimaciones de RU aumentaron con los tratamientos de protección, incrementándose también la digestibilidad intestinal de la materia seca en la HG. Los bajos valores de la digestibilidad de la proteína de GP tratado y sin tratar sugieren la presencia de algún factor antitripsico no termolábil es esta harina. Los tratamientos de protección incrementaron consistentemente la fracción de materia seca y PB digerida intestinalmente en los dos alimentos, mientras que la fracción de almidón en la muestra de GP solamente aumentó numéricamente (60,5% de media). Sin embargo, los tratamientos también redujeron la fermentación de la materia orgánica, lo cual podría disminuir la síntesis de proteína microbiana. Los estudios de electroforesis muestran la práctica desaparición de la albumina por la degradación ruminal en ambos alimentos, así como que los cambios en otras proteínas de la muestra RU fueron más pronunciados en GP que en HG. La composición de las bacterias asociadas con las fases de digesta ruminal sólida (BAS) y líquida (BAL) fue estudiada para revisar la precisión de un sistema de predicción previo que determinaba la infravaloración del aporte de nutrientes correspondiente a las BAS cuando de usa 15N como marcador y las BAL como referencia microbiana (capitulo 4). Al comparar con BAS, BAL mostraron menores contenidos en materia orgánica, polisacáridos de glucosa y lípidos totales y un mayor contenido en PB, así como un mayor enriquecimiento en 15N. Los datos obtenidos en el estudio actual se ajustan bien a la ecuación previa que predice el enriquecimiento en 15N de las BAS a partir del mismo valor en BAL. Esta nueva ecuación permite establecer que se produce una infravaloración de un 22% en el aporte de PB al animal a partir de las BAS sintetizadas si las BAL son usadas como muestras de referencia. Una segunda relación calculada utilizando los valores medios por dieta expuestos en numerosos trabajos encontrados en la literatura confirma la magnitud de este error. Esta infravaloración asociada al uso de BAL como referencia fue mayor para el aporte de glucosa (43,1%) y todavía mayor para el aporte de lípidos (59,9%), como consecuencia de los menores contenidos de ambas fracciones en BAL frente a SAB. Estos errores deberían ser considerados para obtener mayor precisión en la estimación del aporte de nutrientes microbianos y mejorar la nutrición de los rumiantes. En el experimento 2 se realizó un estudio de producción (capitulo 5) para evaluar los efectos del tratamiento de las harinas HG y GP con soluciones de ácido málico o ácido ortofosfórico sobre el crecimiento, el consumo de concentrado y el rendimiento y engrasamiento de las canales de corderos de engorde. Noventa corderos machos de cruce entrefino procedentes de tres granjas comerciales (peso inicial medio = 14,6, 15,3 y 13,3 kg, respectivamente) fueron asignados aleatoriamente a cinco dietas con diferentes niveles de proteína y diferentes tratamientos con ácidos y engordados hasta un peso medio al sacrificio de 25 kg. Las fuentes de proteína en el pienso control (C; PB=18,0%) fueron harina de soja, HG y GP sin tratar. En tres de los piensos experimentales, las harinas tratadas con ácido ortofosfórico sustituyeron a las de HG y GP sin tratar (Control Ortofosfórico, PC; PB=18,0% sobre materia seca), sustituyéndose, además, la harina de soja parcialmente (Sustitución Media Ortofosfórico, MSP; PB=16,7%) o totalmente (Sustitución Total Ortofosfórico, TSP; PB=15,6%). Finalmente, en uno de los piensos el ácido ortofosfórico fue reemplazo por acido málico para proteger ambas harinas (Sustitución Media Málico, MSM; PB= 16,7%). La paja de trigo (fuente de forraje) y el concentrado fueron ofrecidos ad libitum. Dieciocho corderos fueron distribuidos en seis cubículos con tres animales para cada dieta. Los datos fueron analizados según un análisis factorial considerando el peso inicial como covariable y la granja de procedencia como bloque. Los datos de consumo de concentrado y eficiencia de conversión fueron analizados usando el cubículo como unidad experimental, mientras que los datos sobre ganancia media diaria, rendimiento a la canal, grasa dorsal y grasa pélvico renal fueron analizados usando el cordero como unidad experimental. No se encontró ningún efecto asociado con el nivel de PB sobre ninguna variable estudiada. Esto sugiere que usando proteínas protegidas es posible utilizar concentrados con 15,6% de PB (sobre materia seca) disminuyendo así la cantidad de concentrados de proteína vegetal a incluir en los piensos y la calidad de los concentrados proteicos. Los corderos alimentados con la dieta MSM tuvieron mayores ganancias medias diarias (15,2%; P= 0,042), y mejores rendimiento a la canal en caliente (1,3 unidades porcentuales; P= 0,037) que los corderos alimentados con el concentrado MSP. Esto podría ser explicado por los efectos benéficos ruminales del malato o por el mayor efecto de protección conseguido con el ácido málico. ABSTRACT The main objective of this thesis project was to increase the protein efficiency in ruminant diets by using protected protein (sunflower meal and spring pea), and improving the prediction of microbial protein supply. Commercial sunflower meal (SFM) and spring pea (SP) were treated with 4 N solutions (200 mL/kg) of malic acid (268.2 g/L) or orthophosphoric acid (130.6 g/L). Daily, two fractions of 12.5 kg of one of these meals were successively sprayed with the tested acid solution in a concrete mixer using a sprayer. Both fractions were then mixed and allowed to rest for 1 h at room temperature. The blend was then dried in a forced air oven at 120 ºC for 1 h. Then the oven was turned off and the treated material was left in the oven overnight. During the drying process, the material was stirred every 30 min during the first 2 h and then every 60 min for the subsequent 5 h. This process was repeated until the amounts of treated flour needed for the different trials performed. In the first experiment (chapter 3), ruminal and intestinal digestion trials were conducted to study the effects of the application of these acid solutions and heat to protect proteins of SFM and SP against ruminal degradation using three wethers fitted with rumen and duodenum cannulae. The ruminal digestion study was carried out in three experimental periods in which the wethers were successively fed three isoproteic diets including SFM and SP, untreated or treated with malic or orthophosphoric acids. The experimental periods of 21 days included successively: 10 days of diet adaptation, SFM and SP particle ruminal transit study (days 11–14) and ruminal nylon-bag incubations (days 15–21). The meals incubated in each experimental period were those corresponding to the associated diet. Rumen bacteria were labelled from days 11 to 21 by continuous intra-ruminal infusion of a 15N source and the rumen was emptied at the end of in situ incubations in each period to isolate solid adherent bacteria and liquid associate bacteria. The intestinal digestion trial was conducted twenty days after the end of the ruminal studies to eliminate the 15N enrichment in the digesta. The tested samples were composite samples obtained pooling the different ruminally undegraded residues to be representative of the chemical composition of the ruminally undegraded fraction (RU). Wethers were fed the untreated diet to determine the intestinal digestibility of untreated and treated meals using the mobile nylon bag technique. In addition, protein in untreated and treated meals and their 0 h, composite and intestinally undigested samples were extracted and subjected to electrophoresis to determine the digestion site of the different protein fractions. Estimates of the RU and its intestinal digestibility of dry matter, organic matter (only for RU), crude protein (CP) and starch (only in SP) were obtained considering ruminal microbial contamination and particle comminution and outflow rates. When corrected for the microbial contamination taking place in the rumen, estimates of RU and intestinal digestibility decreased in all tested fractions for both feeds. All RU estimates increased with the protective treatments, whereas intestinal digestibility-dry matter also increased in SFM. Low intestinal digestibility-CP values in untreated and treated samples suggested the presence of non-heat labile antitrypsin factors in SP. Protective treatments of both feeds led to consistent increases in the intestinal digested fraction of dry matter and CP, being only numerically different for SP-starch (60.5% as average). However, treatments also reduced the organic matter fermentation, which may decrease ruminal microbial protein synthesis. Electrophoretic studies showed albumin disappearance in both SFM and SP, whereas changes in other RU proteins were more pronounced in SP than SFM. The chemical composition of bacteria associated with solid (SAB) and liquid (LAB) rumen-digesta phases was studied to examine the accuracy of a previous regression system determining the underevaluation of SAB-nutrient supply using 15N as marker and LAB as microbial reference (chapter 4). Compared with SAB, LAB showed lower contents of organic matter, polysaccharide-glucose and total lipids and the opposite for the CP content and the 15N enrichment. Present data fitted well to the previous relationship predicting the 15N enrichment of SAB from the same value in LAB. This new equation allows establishing an underevaluation in the supply of CP from the synthesized SAB in 22.0% if LAB is used as reference. Another relationship calculated using mean diet values from the literature confirmed the magnitude of this error. This underevaluation was higher for the supply of glucose (43.1%) and still higher for the lipid supply (59.9%) as a consequence of the lower contents of these both fractions in LAB than in SAB. These errors should be considered to obtain more accurate estimates of the microbial nutrient supply and to improve ruminant nutrition. A production study was performed in experiment 2 (chapter 5) to examine the effects of treating SFM and SP meals with orthophosphoric or malic acid solutions on growth performance, concentrate intake, and carcass yield and fatness of growing-fattening lambs. Ninety "Entrefino" cross male lambs from three commercial farms (average initial body weights (BW) = 14.6, 15.3 and 13.3 kg) were randomly assigned to five diets with different acid treatment and protein levels, and fattened to an average slaughter weight of 25 kg. Protein sources in the control concentrate (C; CP=18%) were soybean meal and untreated SFM and SP. In three of the experimental concentrates, orthophosphoric acid-treated meals substituted untreated SFM and SP (Orthophosphoric Control, PC; CP=18% dry matter basis), and soybean meal was partially (Medium Substitution Orthophosphoric, MSP; CP=16.7%) or totally removed (Total Substitution Orthophosphoric, TSP; CP=15.6%). In addition, in one concentrate orthophosphoric acid was replaced by malic acid to protect these meals (Medium Substitution Malic, MSM; CP= 16.7%). Wheat straw (roughage source) and concentrate were offered ad libitum. Eighteen lambs were allocated to six pens of three animals on each diet. Data were analyzed using a factorial analysis with initial body weight BW as covariate and farm of origin as block. Data on concentrate intake and feed conversion efficiency were analyzed using pen as experimental unit, while data on average daily gain, carcass yield, dorsal fat, and kidney-pelvic-fat were analyzed with lamb as experimental unit. No effect associated with the CP level was observed on any parameter. This suggests that with protected proteins it is possible to feed concentrates with 15.6% CP (dry matter basis) reducing the quantity of vegetable protein meals to include in the concentrate as well as the quality of the protein concentrates. Lambs feed MSM had higher average daily gains (15.2%; P= 0.042), and better hot carcass yields (1.3 percentage points; P= 0.037) than lambs feed MSP. This probably can be explained by ruminal malate actions and by greater protection effects obtained with malic acid.

Cell adhesion and the integrin-linked kinase regulate the LEF-1 and β-catenin signaling pathways

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that can interact directly with the cytoplasmic domains of the β1 and β3 integrin subunits and whose kinase activity is modulated by cell–extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell–cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. We now show that modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of E-cadherin expression, translocation of β-catenin to the nucleus, formation of a complex between β-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1/β-catenin complex. We also find that LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and that LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-ras or v-src oncogenes do not result in the activation of LEF-1/β-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1/β-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cell–matrix interactions and cell–cell adhesion as well as components of the Wnt signaling pathway.

Glyceraldehyde-3-phosphate dehydrogenase: Nuclear translocation participates in neuronal and nonneuronal cell death

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily reflects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.

Regulation of Bcl-xL expression in human keratinocytes by cell–substratum adhesion and the epidermal growth factor receptor

Cell–substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell–substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-xL. Expression of Bcl-xL protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-xL steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-xL expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-xL represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-xL expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.

The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean

Abscisic acid (ABA), a cleavage product of carotenoids, is involved in stress responses in plants. A well known response of plants to water stress is accumulation of ABA, which is caused by de novo synthesis. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. This step generates the C15 intermediate xanthoxin and C25-apocarotenoids. A cDNA, PvNCED1, was cloned from wilted bean (Phaseolus vulgaris L.) leaves. The 2,398-bp full-length PvNCED1 has an ORF of 615 aa and encodes a 68-kDa protein. The PvNCED1 protein is imported into chloroplasts, where it is associated with the thylakoids. The recombinant protein PvNCED1 catalyzes the cleavage of 9-cis-violaxanthin and 9′-cis-neoxanthin, so that the enzyme is referred to as 9-cis-epoxycarotenoid dioxygenase. When detached bean leaves were water stressed, ABA accumulation was preceded by large increases in PvNCED1 mRNA and protein levels. Conversely, rehydration of stressed leaves caused a rapid decrease in PvNCED1 mRNA, protein, and ABA levels. In bean roots, a similar correlation among PvNCED1 mRNA, protein, and ABA levels was observed. However, the ABA content was much less than in leaves, presumably because of the much smaller carotenoid precursor pool in roots than in leaves. At 7°C, PvNCED1 mRNA and ABA were slowly induced by water stress, but, at 2°C, neither accumulated. The results provide evidence that drought-induced ABA biosynthesis is regulated by the 9-cis-epoxycarotenoid cleavage reaction and that this reaction takes place in the thylakoids, where the carotenoid substrate is located.

Dynamic regulation of c-Jun N-terminal kinase activity in mouse brain by environmental stimuli

Activation of the recently identified c-Jun N-terminal kinases (JNKs) typically results in programmed cell death (apoptosis) in neurons and other cell types grown in culture. However, the effects of JNK activation in the central nervous system in vivo are unknown. At baseline, JNK activity in mice was on average 17-fold higher in brain than in peripheral organs, whereas JNK protein levels were similar. In brain, JNK was expressed primarily in neurons. Restraining mice or allowing them to explore a novel environment rapidly increased JNK activity 3- to 15-fold in various brain regions, but these manipulations did not increase brain activity of the extracellular signal-regulated kinase. Because noninvasive environmental stimuli that do not induce neurodegeneration elicited prominent increases in JNK activity in the brain, we conclude that acute activation of the JNK cascade in central nervous system neurons does not induce neuronal apoptosis in vivo. In contrast, the high baseline activity of JNK in the brain and the activation of the JNK cascade by environmental stimuli suggest that this kinase may play an important physiological role in neuronal function.

Temporary Disruption of the Plasma Membrane Is Required for c-fos Expression in Response to Mechanical Stress

Mechanically stressed cells display increased levels of fos message and protein. Although the intracellular signaling pathways responsible for FOS induction have been extensively characterized, we still do not understand the nature of the primary cell mechanotransduction event responsible for converting an externally acting mechanical stressor into an intracellular signal cascade. We now report that plasma membrane disruption (PMD) is quantitatively correlated on a cell-by-cell basis with fos protein levels expressed in mechanically injured monolayers. When the population of PMD-affected cells in injured monolayers was selectively prevented from responding to the injury, the fos response was completely ablated, demonstrating that PMD is a requisite event. This PMD-dependent expression of fos protein did not require cell exposure to cues inherent in release from cell–cell contact inhibition or presented by denuded substratum, because it also occurred in subconfluent monolayers. Fos expression also could not be explained by factors released through PMD, because cell injury conditioned medium failed to elicit fos expression. Translocation of the transcription factor NF-κB into the nucleus may also be regulated by PMD, based on a quantitative correlation similar to that found with fos. We propose that PMD, by allowing a flux of normally impermeant molecules across the plasma membrane, mediates a previously unrecognized form of cell mechanotransduction. PMD may thereby lead to cell growth or hypertrophy responses such as those that are present normally in mechanically stressed skeletal muscle and pathologically in the cardiovascular system.

RhoA GTPase and Serum Response Factor Control Selectively the Expression of MyoD without Affecting Myf5 in Mouse Myoblasts

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

Ex vivo evaluation of a Taylor-Couette flow, immobilized heparinase I device for clinical application

Efficient and safe heparin anticoagulation has remained a problem for continuous renal replacement therapies and intermittent hemodialysis for patients with acute renal failure. To make heparin therapy safer for the patient with acute renal failure at high risk of bleeding, we have proposed regional heparinization of the circuit via an immobilized heparinase I filter. This study tested a device based on Taylor-Couette flow and simultaneous separation/reaction for efficacy and safety of heparin removal in a sheep model. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. The device, referred to as a vortex flow plasmapheretic reactor, consisted of two concentric cylinders, a priming volume of 45 ml, a microporous membrane for plasma separation, and an outer compartment where the immobilized heparinase I was fluidized separately from the blood cells. Manual white cell and platelet counts, hematocrit, total protein, and fibrinogen assays were performed. Heparin levels were indirectly measured via whole-blood recalcification times (WBRTs). The vortex flow plasmapheretic reactor maintained significantly higher heparin levels in the extracorporeal circuit than in the sheep (device inlet WBRTs were 1.5 times the device outlet WBRTs) with no hemolysis. The reactor treatment did not effect any physiologically significant changes in complete blood cell counts, platelets, and protein levels for up to 2 hr of operation. Furthermore, gross necropsy and histopathology did not show any significant abnormalities in the kidney, liver, heart, brain, and spleen.

Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5′ leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.