Neurophysiologic and proteomic investigations of experience-dependent plasticity in the somatosensory cortex of the adult mouse following chronic whisker stimulation

RESUME Les follicules des vibrisses des rongeurs sont représentés sous la forme d'une carte topographique dans le cortex à tonneaux. Lorsque un groupe de vibrisses est coupé pendant plusieurs jours chez un rongeur adulte, en laissant les autres vibrisses intactes, le champ réceptif des neurones du cortex à tonneaux est modifié, ce qui démontre que les cartes corticales sont plastiques. Dans notre étude, une expérience sensorielle a été induite chez une souris adulte se comportant librement en stimulant chroniquement une de ses vibrisses pendant 24h. Par une analyse des potentiels de champ locaux, nous démontrons que les caractéristiques spatiotemporelles du flux d'excitation évoqué par la vibrisse principale (VP) dans la colonne corticale correspondante à la vibrisse stimulée n'est pas altéré. Par contre, l'enregistrement des potentiels d'actions d'un total de 1041 neurones à travers le cortex à tonneaux révèlent plusieurs modifications de l'activité neuronale. L'activité spontanée ainsi que la réponse évoquée par la VP sont déprimées dans la colonne corticale stimulée (nombre moyen de potentiels d'action évoqués par la VP diminue de 25 % et 36% dans la couche IV et les couches II&III). La réponse des neurones à la vibrisse stimulée diminue également dans les colonnes corticales adjacentes, «non-stimulées». La dépression de l'activité spontanée et de la réponse à la VP est localisée à la colonne corticale stimulée. Dans le tonneau stimulé, la première partie de la réponse à la VP n'est pas affaiblie, démontrant que la dépression de la réponse n'est pas due à un phénomène de plasticité sous-corticale ou thalamocorticale. La stimulation chronique d'une vibrisse entraîne une augmentation du nombre de synapses GABAergiques dans la couche IV du tonneau correspondant (Knott et al, 2002). Dès lors, nos résultats suggèrent qu'une augmentation de l'inhibition dans le tonneau stimulé serait à l'origine de la diminution des potentiels d'action évoqués par la vibrisse stimulée et en conséquence de l'amplitude du flux d'excitation vers les couches II&III puis vers les colonnes corticales adjacentes. Toutes les réponses des neurones du tonneau stimulé ne sont pas déprimées. Les réponses des neurones à la vibrisse voisine caudale à VP diminuent dans la couche IV (42%) et dans les couches II&III (52%) mais pas les réponses aux 7 autres vibrisses voisines. Les entrées synaptiques en provenance de la vibrisse caudale pourraient avoir été spécifiquement déprimées en raison d'une décorrélation prolongée entre l'activité évoquée dans les chemins sensoriels relatifs à la vibrisse stimulée et à la vibrisse caudale, spécificité qui découlerait du fait que, parmi les vibrisses voisines à la VP, la vibrisse caudale génère les réponses les plus fortes dans la colonne corticale. Quatre jours après l'arrêt de la stimulation, l'activité neuronale n'est plus déprimée; au contraire, nous observons une potentiation des réponses à la VP dans la couche IV de la colonne corticale stimulée. De plus, nous montrons que l'expression des protéines GLT-1 et GLAST, deux transporteurs astrocytaires du glutamate, est augmentée de ~2.5 fois dans la colonne corticale stimulée, indiquant l'existence d'une «plasticité gliale» et suggérant que les cellules gliales participent activement à l'adaptation du cerveau à l'expérience. ABSTRACT In the barrel cortex, mystacial whisker follicles are represented in the form of a topographie map. The selective removal of a set of whiskers while sparing others for several days in an adult rodent alters receptive field of barrel cortex neurons, demonstrating experience-dependent plasticity of cortical maps. Here sensory experience was altered by chronic stimulation of a whisker for a 24h period in a freely behaving adult mouse. By means of an evoked local field potential analysis, we show that chronic stimulation does not alter the flow of excitation evoked by the principal whisker (PW) in the stimulated barrel column. However, the recording of neuronal firing from a total of 1041 single units throughout the barrel cortex reveals several changes in neuronal activity. Immediately after chronic stimulation, spontaneous activity as well as PW-responses are depressed in the stimulated barrel column (mean number of spikes per PW-deflection decreases by 25% and 36% in layer IV and layers II&III, respectively). Neuronal responses towards the chronically stimulated whisker are also significantly depressed in layers II&III of the adjacent "non-stimulated" barrel' columns. The depression of both spontaneous activity and PW-responses are restricted to the stimulated ban-el column. The earliest time epoch of the PW-response in the stimulated barrel is not depressed, demonstrating that the decrease of cortical responses is not due to subcortical or thalamocortical plasticity. The depression of PW-response in the stimulated barrel correlates with an increase in the number of GABAergic synapses in layer IV (Knott et al., 2002). Therefore, our results suggest that an increase in inhibition within the stimulated barrel may reduce its excitatory output and accordingly the flow of excitation towards layers and the subsequent horizontal spread into adjacent barrel columns. Not all responses of neurons in the stimulated barrel are depressed. Neuronal responses towards the caudal in-row whisker decrease by 42% in layer IV and 52% in layers MM but responses to the other 7 immediate surround whiskers (SWs) are not affected. The synaptic inputs from the SW that elicit the strongest responses in the stimulated barrel may have been specifically depressed following a prolonged period of diminished coherence between neuronal activity evoked in the pathways from the chronically stimulated whisker and from its surrounding in-row whisker. Four days after the cessation of the stimulation, depression of neuronal activity is no longer present; on the contrary, we observe a small but significant potentiation of PW-responses in layer IV of the stimulated barrel column. Moreover we show that the expression of astrocytic glutamate transporters GLT-1 and GLAST proteins were both upregulated by ~2.5 fold in the stimulated barrel column, which indicates that glial cells exhibit experience-dependent functional changes and could actively take part in the adaptation of the cerebral cortex to experience.

Assessing changes in Cd phytoavailability to tomato in amended calcareous soils.

A plot study was conducted to assess changes in Cd phytoavailability to a tomato cultivar in an agricultural soil in Southeastern Spain amended in two different ways (A and B), under controlled conditions. The experimental soil corresponded to a fine-loamy carbonatic thermic Calcidic Haploxeroll (Soil Survey Staff, 1998). A) Soil was amended with a single application of sewage sludge from a municipal source that had a total Cd concentration of 0.5 mg kg-1 at a rate that represented a final average concentration in the mixture of soil and sludge of less than 50 µg Cd kg-1. B) The amendment consisted of the addition of a mineral fertiliser with the same amount of NPK as in the sewage sludge application. The final levels of Cd were supposed to be negligible. A plot series without amendments was also performed (C). DTPA plus triethanolamine, and ammonium acetate extractable fractions in soils were analysed for all the plots. The time-dependent Cd accumulation in different parts of the tomato plants was studied by means of a Cd salt treatment. For each block (A, B, and C) four levels of Cd (0, 3, 30, 100 mg kg-1) were added as CdCl2. There was a significant increase in plant Cd after the initial cropping. Tomato stems, leaves and fruits were analysed separately for Cd determination. Differential Cd distribution and accumulation in tomato parts was detected.

Botulinum neurotoxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release,was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods.To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.

Environmental control of the Pom1-dependent cell-size regulation pathway in fission yeast

Cells couple their growth and division rate in response to nutrient availability to maintain a constant size. This co-ordination happens either at the G1-S or the G2-M transition of the cell cycle. In the rod-shaped fission yeast, size regulation happens at the G2-M transition prior to mitotic commitment. Recent studies have focused on the role of the DYRK-family protein kinase Pom1, which forms gradients emanating from cell poles and inhibits the mitotic activator kinase Cdr2, present at the cell middle. Pom1 was proposed to inhibit Cdr2 until cells reached a critical size before division. However when and where Pom1 inhibits Cdr2 is not clear as medial Pom1 levels do not change during cell elongation. Here I show that Pom1 gradients are susceptible to environmental changes in glucose. Specifically, upon glucose limitation, Pom1 re-localizes from the poles to the cell sides where it delays mitosis through regulating Cdr2. This re-localization occurs due to microtubule de- stabilization and lateral catastrophes leading to transient deposition of the Pom1 gradient nucleator Tea4 along the cell cortex. As Tea4 localization to cell sides is sufficient to recruit Pom1, this explains the mechanism of Pom1 re-localization. Microtubule destabilization and consequently Tea4 and Pom1 spread depends on the activity of the cAMP-dependent Protein Kinase A (PKA/Pka1), as pka1 mutant cells have stable microtubules and retain polar Tea4 and Pom1 under limited glucose. PKA signaling negatively regulates the microtubule rescue factor CLASP/Cls1, thus reducing its ability to stabilize microtubules. Thus PKA signaling tunes CLASP activity to promote microtubule de-stabilization and Pom1 re-localization upon glucose limitation. I show that the side-localized Pom1 delays mitosis and balances the role of the mitosis promoting, mitogen-associated protein kinase (MAPK) protein Sty1. Thus Pom1 re-localization may serve to buffer cell size upon glucose limitation. -- Afin de maintenir une taille constante, les cellules régulent leur croissance ainsi que leur taux de division selon les nutriments disponibles dans le milieu. Dans la levure fissipare, cette régulation de la taille précède l'engagement mitotique et se fait à la transition entre les phases G2 à M du cycle cellulaire. Des études récentes se sont focalisées sur le rôle de la protéine Pom1, membre de la famille des DYRK kinase. Celle-ci forme un gradient provenant des pôles de la cellule et inhibe l'activateur mitotique Cdr2 présent au centre de la cellule. Le model propose que Pom1 inhibe Cdr2 jusqu'à atteindre une taille critique avant la division. Cependant quand et à quel endroit dans la cellulle Pom1 inhibe Cdr2 n'était pas clair car les niveaux médians de Pom1 ne changent pas au cours de la l'élongation des cellules. Dans cette étude, je montre que les gradients de Pom1 sont sensibles aux changements environnementaux du taux de glucose. Plus spécifiquement, en conditions limitantes de glucose, Pom1 se relocalise des pôles de la cellule pour se distribuer sur les côtés de celle-ci. Par conséquent, un délai d'entrée en mitose est observé dû à l'inhibition Cdr2 par Pom1. Cette délocalisation est due à la déstabilisation des microtubules qui va conduire à une déposition transitoire de Tea4, le nucléateur du gradient de Pom1, tout au long du cortex de la cellule. Comme la localisation de Tea4 sur les côtés de la cellule est suffisante pour recruter la protéine Pom1, ceci explique le mécanisme de relocalisation de celle-ci. La déstabilisation des microtubules et par conséquent la diffusion de Tea4 et Pom1 dépendent de l'activité de la protéine kinase A dépendante de l'AMP cyclique (PKA/Pka1). En absence de pka1, la stabilité des microtubules n'est pas affectée ce qui permet la rétention de Tea4 et Pom1 aux pôles de la cellule même en conditions limitantes de glucose. La signalisation via PKA régule négativement le facteur de sauvetage des microtubules CLASP/Cls1 et permet donc de réduire sa fonction de déstabilisation des microtubules. Ainsi la signalisation via PKA affine l'activité des CLASP pour promouvoir la déstabilisation des microtubules et la relocalisation de Pom1 en conditions limitantes de glucose. Je montre que la localisation sur les côtés retarde l'entrée en mitose et compense l'action de la protéine Sty1, connue pour être une MAPK qui induit l'entrée en mitose. Ainsi, la relocalisation de Pom1 pourrait servir à tamponner la taille de la cellule en condition limitantes de glucose. -- Various cell types in the environment such as bacterial, plant or animal cells have a distinct cellular size. Maintaining a constant cell size is important for fitness in unicellular organisms and for diverse functions in multicellular organisms. Cells regulate their size by coordinating their growth rate to their division rate. This coupling is important otherwise cells would get progressively smaller or larger after each successive cell cycle. In their natural environment cells may face fluctuations in the available nutrient supply. Thus cells have to coordinate their division rate to the variable growth rates shown under different nutrient conditions. During my PhD, I worked with a single-celled rod shaped yeast called the fission yeast. These cells are longer when the nutrient supply is abundant and shorter when the nutrient supply is scarce. A protein that senses changes in the external carbon source (glucose) is called Protein Kinase A (PKA). The rod shape of fission yeast cells is maintained thanks to a structural backbone called the cytoskeleton. One of the components of this backbone is called microtubules, which are small tube like structures spanning the length of the cell. They transport a protein called Tea4, which in turn is important for the proper localization of another protein Pom1 to the cell ends. Pom1 helps to maintain proper shape and size of these rod shaped yeast cells. My thesis work showed that upon reduction in the external nutrient (glucose) levels, microtubules become less stable and show an alteration in their organization. A significant percentage of the microtubules contact the side of the cell instead of touching only the cell tip. This leads to the spreading of the protein Pom1 away from the tips all around the cell periphery. This helps fission yeast cells to maintain the proper size required under these conditions of limited glucose supply. I further showed that the protein PKA regulates microtubule stability and organization and thus Pom1 spreading and maintenance of proper cell size. Thus my work led to the discovery of a novel pathway by which fission yeast cells maintain their size under limited supply of glucose. -- Divers types cellulaires dans l'environnement tels que les bactéries, les plantes ou les cellules animales ont une taille précise. Le maintien d'une taille cellulaire constante est importante pour le fitness des organismes unicellulaire ainsi que pour multiples fonctions dans les organismes multicellulaires. Les cellules régulent leur taille en coordonnant le taux de croissance avec le taux de division. Ce couplage est essentiel sinon les cellules deviendraient progressivement plus petites ou plus grandes après chaque cycle cellulaire. Dans leur habitat naturels les cellules peuvent faire face a des fluctuations dans le taux de nutriment disponible. Les cellules doivent donc coordonner leur taux de division aux taux variables de croissances perçus dans les différentes conditions nutritionnels. Pendant ma thèse, j'ai travaillée sur une levure unicellulaire, en forme de bâtonnet, nommé levure fissipare ou levure de fission. La taille de ces cellules est plus grande quand le taux de nutriments est grand et plus courte quand celui-ci est plus faible. Une protéine qui perçoit les changements dans le taux externe de la source de carbone (glucose) est nommée PKA pour protéine kinase A. La forme en bâtonnet de la cellule est due aux caractères structuraux du cytosquelette. Une composante importante de ce cytosquelette sont les microtubules, dont la structures ressemble à des petit tubes qui vont d'un bout à l'autre de la cellule. Ces microtubules transportent une protéine importante nommée Tea4 qui à leur tour importante pour la bonne localisation d'une autre protéine Pom1 aux extrémités de la cellule. La protéine Pom1 aide à maintenir la taille appropriée des levures fissipares. Mon travail de thèse a montré qu'en présence de taux faible de nutriments (glucose) les microtubules deviennent de moins en moins stables et montrent une désorganisation globale. Un pourcentage significatif des microtubules touche les côtés de la cellule aux lieu d'atteindre uniquement les extrémités. Ceci a pour conséquence une diffusion de Pom1 tout au long du cortex de la cellule. Ceci aide les levures fissipares à maintenir la taille appropriée pendant ce stress nutritionnel. De plus, je montre que PKA régule la stabilité et l'organisation des microtubules et par conséquent la diffusion de Pom1 et le maintien d'une taille constante. En conclusion, mon travail a conduit à la découverte d'un nouveau mécanisme par lequel la levure fissipare maintient sa taille dans des conditions limitantes en glucose.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Fish colonization of artificial reefs in a large Neotropical reservoir: material type and successional changes

Artificial reefs have barely been used in Neotropical reservoirs (about five studies in three reservoirs), despite their potential as a fishery management tool to create new habitats and also to understand fish ecology. We experimentally assessed how reef material (ceramic, concrete, and PVC) and time modulated fish colonization of artificial reefs deployed in Itaipu Reservoir, a large reservoir of the mainstem Parana´ River, Brazil. Fish richness, abundance, and biomass were significantly greater in the reef treatments than at control sites. Among the experimental reefs, ceramic followed by the concrete treatments were the materials most effectively colonized, harboring the majority of the 13 fish species recorded. Although dependent on material type, many of the regularities of ecological successions were also observed in the artificial reefs, including decelerating increases in species richness, abundance, mean individual size, and species loss rates with time and decelerating decreases of species gain and turnover rates. Species composition also varied with material type and time, together with suites of life history traits: more equilibrium species (i.e., fishes of intermediate size that often exhibit parental care and produce fewer but larger offspring) of the Winemiller-Rose model of fish life histories prevailed in later successional stages. Overall, our study suggests that experimental reefs are a promising tool to understand ecological succession of fish assemblages, particularly in tropical ecosystems given their high species richness and low seasonality

Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hall- mark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM (the calcein acetoxymethyl-ester) on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phos-phate pathway was significantly altered. To elucidate whe-ther these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

Experimental poisoning by Senecio brasiliensis in calves: quantitative and semi-quantitative study on changes in the hepatic extracellular matrix and sinusoidal cells

Extracellular matrix plays an important role in chronic hepatic lesions and has been studied in experimental intoxication models. However in cattle, studies on chronic disease have focused on the hepatocellular damage and extracellular matrix (ECM) changes are usually overlooked. There are no specific studies on the hepatic ECM in either normal or chronically damaged bovine liver. Thus an experimental model of hepatic toxicity model using Senecio brasiliensis poisoned calves was designed. Senecio brasiliensis contains pyrrolizidine alkaloids which cause either acute or chronic progressive dose dependent liver damage. Five calves were orally fed with 0.38g of dry leaves of S. brasiliensis/kg/day for 24 days. Liver needle biopsy specimens were obtained every 15 days for 60 days. Clinical signs of digestive complications appeared at 3rd week. One calf died on 45th day and four were evaluated up to 60th day. Biopsy samples were processed for routine light microscopy, immuno-histochemistry and transmission electron microscopy. From 30th day on progressive liver damage characterized by hepatocellular ballooning, necrosis, apoptosis and megalocytosis, centrilobular, pericellular and portal fibrosis were seen by light microscopy. Quantitative and semi-quantitative measurements of hepatic ECM components were performed before and after the onset of lesions. Morphometric analysis of total collagen and elastic fiber system was conducted. Total collagen and I and III collagen types progressively increased in throughout the liver of affected calves. Changes in location, amount and disposition of the elastic fiber system were also observed. Then numbers of Kupffer cells were significantly increased at 30th day and total numbers of sinusoidal cells were significantly increased at 45th and 60th days. Liver damage was progressive and irreversible even after the exposure to the plant was discontinued. Severe fibrotic lesions occurred mainly in portal tracts, followed by veno-occlusive and pericellular fibrosis. Collagen types I and III s were present in every normal and damaged liver, with predominance of type I. In affected calves the increase of total collagen and elastic fibers system paralleled the number of total sinusoidal cells.

Disturbance and ecosystem functioning : the role of changes in benthic biological traits

Rapid changes in biodiversity are occurring globally, as a consequence of anthropogenic disturbance. This has raised concerns, since biodiversity is known to significantly contribute to ecosystem functions and services. Marine benthic communities participate in numerous functions provided by soft-sedimentary ecosystems. Eutrophication-induced oxygen deficiency is a growing threat against infaunal communities, both in open sea areas and in coastal zones. There is thus a need to understand how such disturbance affects benthic communities, and what is lost in terms of ecosystem functioning if benthic communities are harmed. In this thesis, the status of benthic biodiversity was assessed for the open Baltic Sea, a system severely affected by broad-scale hypoxia. Long-term monitoring data made it possible to establish quantitative biodiversity baselines against which change could be compared. The findings show that benthic biodiversity is currently severely impaired in large areas of the open Baltic Sea, from the Bornholm Basin to the Gulf of Finland. The observed reduction in biodiversity indicates that benthic communities are structurally and functionally impoverished in several of the sub-basins due to the hypoxic stress. A more detailed examination of disturbance impacts (through field studies and -experiments) on benthic communities in coastal areas showed that changes in benthic community structure and function took place well before species were lost from the system. The degradation of benthic community structure and function was directed by the type of disturbance, and its specific temporal and spatial characteristics. The observed shifts in benthic trait composition were primarily the result of reductions in species’ abundances, or of changes in demographic characteristics, such as the loss of large, adult bivalves. Reduction in community functions was expressed as declines in the benthic bioturbation potential and in secondary biomass production. The benthic communities and their degradation accounted for a substantial proportion of the changes observed in ecosystem multifunctionality. Individual ecosystem functions (i.e. measures of sediment ecosystem metabolism, elemental cycling, biomass production, organic matter transformation and physical structuring) were observed to differ in their response to increasing hypoxic disturbance. Interestingly, the results suggested that an impairment of ecosystem functioning could be detected at an earlier stage if multiple functions were considered. Importantly, the findings indicate that even small-scale hypoxic disturbance can reduce the buffering capacity of sedimentary ecosystem, and increase the susceptibility of the system towards further stress. Although the results of the individual papers are context-dependent, their combined outcome implies that healthy benthic communities are important for sustaining overall ecosystem functioning as well as ecosystem resilience in the Baltic Sea.

Effects of bromocriptine on serum prolactin levels, pituitary weight and immunoreactive prolactin cells in estradiol-treated ovariectomized rats: an experimental model of estrogen-dependent hyperprolactinemia

The present study was designed to assess the effects of bromocriptine, a dopamine agonist, on pituitary wet weight, number of immunoreactive prolactin cells and serum prolactin concentrations in estradiol-treated rats. Ovariectomized Wistar rats were injected subcutaneously with sunflower oil vehicle or estradiol valerate (50 or 300 µg rat-1 week-1) for 2, 4 or 10 weeks. Bromocriptine (0.2 or 0.6 mg rat-1 day-1) was injected daily during the last 5 or 12 days of estrogen treatment. Data were compared with those obtained for intact control rats. Administration of both doses of estrogen increased serum prolactin levels. No difference in the number of prolactin cells in rats treated with 50 µg estradiol valerate was observed compared to intact adult animals. In contrast, rats treated with 300 µg estradiol valerate showed a significant increase in the number of prolactin cells (P<0.05). Therefore, the increase in serum prolactin levels observed in rats treated with 50 µg estradiol valerate, in the absence of morphological changes in the pituitary cells, suggests a "functional" estrogen-induced hyperprolactinemia. Bromocriptine decreased prolactin levels in all estrogen-treated rats. The administration of this drug to rats previously treated with 300 µg estradiol valerate also resulted in a significant decrease in pituitary weight and number of prolactin cells when compared to the group treated with estradiol alone. The general antiprolactinemic and antiproliferative pituitary effects of bromocriptine treatment reported here validate the experimental model of estrogen-induced hyperprolactinemic rats

Circadian time-dependent effects of fencamfamine on inhibition of dopamine uptake and release in rat striatal slices

Fencamfamine (FCF) is a central stimulant that facilitates central dopaminergic transmission through inhibition of dopamine uptake and enhanced release of the transmitter. We evaluated the changes in the inhibition of uptake and the release of striatal [3H]-dopamine at 9:00 and 21:00 h, times corresponding to maximal and minimal behavioral responses to FCF, respectively. Adult male Wistar rats (200-250 g) maintained on a 12-h light/12-h dark cycle (lights on at 7:00 h) were used. In the behavioral experiments the rats (N = 8 for each group) received FCF (3.5 mg/kg, ip) or saline at 9:00 or 21:00 h. Fifteen minutes after treatment the duration of activity (sniffing, rearing and locomotion) was recorded for 120 min. The basal motor activity was higher (28.6 ± 4.2 vs 8.4 ± 3.5 s) after saline administration at 21:00 h than at 9:00 h. FCF at a single dose significantly enhanced the basal motor activity (38.3 ± 4.5 vs 8.4 ± 3.5 s) and increased the duration of exploratory activity (38.3 ± 4.5 vs 32.1 ± 4.6 s) during the light, but not the dark phase. Two other groups of rats (N = 6 for each group) were decapitated at 9:00 and 21:00 h and striata were dissected for dopamine uptake and release assays. The inhibition of uptake and release of [3H]-dopamine were higher at 9:00 than at 21:00 h, suggesting that uptake inhibition and the release properties of FCF undergo daily variation. These data suggest that the circadian time-dependent effects of FCF might be related to a higher susceptibility of dopamine presynaptic terminals to the action of FCF during the light phase which corresponds to the rats' resting period

Abnormal nocturnal blood pressure fall in normotensive adolescents with insulin-dependent diabetes is ameliorated following glycemic improvement

Lack of the physiological nocturnal fall in blood pressure (BP) has been found in diabetics and it seems to be related to the presence of diabetic complications. The present study examined the changes in the nocturnal BP pattern of 8 normotensive insulin-dependent diabetic adolescents without nephropathy following improvement in glycemic control induced by an 8-day program of adequate diet and exercise. The same number of age- and sex-matched control subjects were studied. During the first and eighth nights of the program, BP was obtained by ambulatory BP monitoring. After a 10-min rest, 3 BP and heart rate (HR) recordings were taken and the mean values were considered to represent their awake values. The monitor was programmed to cuff insufflation every 20 min from 10:00 p.m. to 7:00 a.m. The glycemic control of diabetics improved since glycemia (212.0 ± 91.5 to 140.2 ± 69.1 mg/dl, P<0.03), urine glucose (12.7 ± 11.8 to 8.6 ± 6.4 g/24 h, P = 0.08) and insulin dose (31.1 ± 7.7 to 16.1 ± 9.7 U/day, P<0.01) were reduced on the last day. The mean BP of control subjects markedly decreased during the sleeping hours of night 1 (92.3 ± 6.4 to 78.1 ± 5.0 mmHg, P<0.001) and night 8 (87.3 ± 6.7 to 76.9 ± 3.6 mmHg, P<0.001). Diabetic patients showed a slight decrease in mean BP during the first night. However, the fall in BP during the nocturnal period increased significantly on the eighth night. The average awake-sleep BP variation was significantly higher at the end of the study (4.2 vs 10.3%, P<0.05) and this ratio turned out to be similar to that found in the control group (10.3 vs 16.3%). HR variation also increased on the eighth night in the diabetics. Following the metabolic improvement obtained at the end of the period, the nocturnal BP variation of diabetics was close to the normal pattern. We suggest that amelioration of glycemic control may influence the awake-sleep BP and HR differences. This effect may be due at least in part to an attenuated insulin stimulation of sympathetic activity

Modulation of staphylokinase-dependent plasminogen activation by mono- and divalent ions

The effect of several ions (Cl-, Na+, K+, Ca2+) on the rate of plasminogen (Pg) activation by recombinant staphylokinase (rSTA) is reported. Both monovalent and divalent ions affect the rate at which Pg is activated by rSTA, in a concentration-dependent manner (range 0-100 mM). In almost all cases, a decrease of the initial velocity of activation was observed. Cl- showed the most striking inhibitory effect at low concentrations (64% at 10 mM). However, in the presence of a fibrin surface, this inhibition was attenuated to 38%. Surprisingly, 10 mM Ca2+ enhanced the Pg activation rate 21% when a polymerized fibrin matrix was present. These data support the idea that ions can modulate the rate of Pg activation through a mechanism that may be associated with changes in the molecular conformation of the zymogen. This effect is strongly dependent on the presence of a fibrin clot.

Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 µM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrolactone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 110- 90- and 50-kDa proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular protein bands was associated with the marked accumulation of two proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more proteins. These findings link the decrease of cyclin-dependent kinase activities to the increase of protease activities within the growth arrest and apoptosis pathways induced by TNF.

A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.