Eficiencia de diferentes cadenas de tratamiento en la eliminación de algas y sus metabolitos

Estudio que analiza dos tipos de tratamiento de algas, ambos basados en filtración, cloración y post-ozonización, ante la posibilidad de que la cantidad de cloro utilizada sobrepase las recomendaciones de la OMS y afecte el sabor y olor del agua tratada.Incluye resultados del estudio, figuras y tablas

Chemical reactivity in matrix-assisted laser desorption/ionization mass spectrometry

During the control of a multistep organic synthesis on a soluble polymer (PEG) by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a chemical reactivity was encountered when the matrix was acidic, for the samples where the amino moiety of the anchored compounds was protected as a Schiff base. Such imine hydrolysis was proven to be solely mediated by the acidic matrix during analyses since the expected protected structures were detected when the experiments were duplicated with a non-acidic matrix. Even if MALDI mass spectrometry was found to be more convenient than electrospray ionization mass spectrometry for the monitoring of liquid phase organic syntheses, the chemical reactivity imparted by the use of a matrix must be taken into account to avoid erroneous spectra interpretations. Copyright 1999 John Wiley & Sons, Ltd.

Substrate recognition and selectivity of peptide deformylase. Similarities and differences with metzincins and thermolysin.

The substrate specificity of Escherichia coli peptide deformylase was investigated by measuring the efficiency of the enzyme to cleave formyl- peptides of the general formula Fo-Xaa-Yaa-NH2, where Xaa represents a set of 27 natural and unusual amino acids and Yaa corresponds to a set of 19 natural amino acids. Substrates with bulky hydrophobic side-chains at the P1' position were the most efficiently cleaved, with catalytic efficiencies greater by two to five orders of magnitude than those associated with polar or charged amino acid side-chains. Among hydrophobic side-chains, linear alkyl groups were preferred at the P1' position, as compared to aryl-alkyl side-chains. Interestingly, in the linear alkyl substituent series, with the exception of norleucine, deformylase exhibits a preference for the substrate containing Met in the P1' position. Next, the influence in catalysis of the second side-chain was studied after synthesis of 20 compounds of the formula Fo-Nle-Yaa-NH2. Their deformylation rates varied within a range of only one order of magnitude. A 3D model of the interaction of PDF with an inhibitor was then constructed and revealed indeed the occurrence of a deep and hydrophobic S1' pocket as well as the absence of a true S2' pocket. These analyses pointed out a set of possible interactions between deformylase and its substrates, which could be the ground driving substrate specificity. The validity of this enzyme:substrate docking was further probed with the help of a set of site-directed variants of the enzyme. From this, the importance of residues at the bottom of the S1' pocket (Ile128 and Leu125) as well as the hydrogen bond network that the main chain of the substrate makes with the enzyme were revealed. Based on the numerous homologies that deformylase displays with thermolysin and metzincins, a mechanism of enzyme:substrate recognition and hydrolysis could finally be proposed. Specific features of PDF with respect to other members of the enzymes with motif HEXXH are discussed.

Striatal target-induced axonal branching of dopaminergic mesencephalic neurons in culture via diffusible factors.

The effects of striatal target cells on the morphological development of dopaminergic neurons were studied in dissociated cultures of embryonic rat mesencephalon. Mesencephalic neurons were cultured for four days in presence of target striatal cells or non target cerebellar ones. The outgrowth of dopaminergic neurons, visualized after tyrosine hydroxylase immunohistochemistry, was examined by quantitative morphometry. In cocultures, the increased complexity of dopaminergic neurites (branching) was the most striking pattern. It was dependent on the presence of target striatal cells as compared to non target ones. Cultures raised in presence or absence of serum lead to suggest the implication of striatal neurons rather than glia. Using MAP2 and phosphorylated neurofilaments immunohistochemistry in combination with tyrosine hydroxylase immunolabelling, it could be shown that the target-induced branching effect concerned only axonal and not dendritic processes. To further define whether diffusible factors from the striatal target would participate in the axonal branching effect, mesencephalic cells were cultured in conditioned medium from striatal neurons. Striatal conditioned medium enhanced dopamine uptake and dopamine neuron branching to the same extent as that observed in striatal cocultures. These findings demonstrate that soluble factors secreted by striatal neurons themselves selectively influence the branching of dopaminergic axons in vitro.

Normal distribution of alpha 2-adrenoceptors in the rat spinal cord and its modification after noradrenergic denervation: a quantitative autoradiographic study.

The distribution of alpha 2 (alpha 2)-adrenoceptors along cervical, thoracic, lumbar, and sacral segments of the spinal cord of normal rats has been studied by quantitative autoradiography using the specific alpha 2-antagonist [3H]rauwolscine as a ligand. In addition, the influence of noradrenergic (NA) denervation [obtained either by complete transection of the spinal cord at vertebrae level T8-T9 or by selective lesion of NA spinal cord system carried out by intracisternal injection of 6-hydroxydopamine (6-OHDA)] on eventual variations of alpha 2-adrenoceptor density at spinal cord target cells was studied in parallel. In control rats, the quantitative analysis of alpha 2-adrenoceptor densities revealed the presence of these receptors throughout the whole gray matter with a preferential location in the superficial dorsal horn. This pattern was the same at all rostro-caudal levels of the cord and appeared very well correlated with the distribution of NA terminals revealed by immunohistochemistry, particularly in the superficial layers of the dorsal horn. After total transection of the spinal cord (caudally to the section) and 6-OHDA-induced lesion, an increase of alpha 2-adrenoceptor density was mainly observed within the distal dorsal horn thus evidencing supersensitivity in this area, while modifications were not detectable in other regions of the spinal gray matter, except at the lumbar level where other dorsal, central, and intermediate zones were significantly enriched.

Modeling the integrative properties of dendrites: application to the striatal spiny neuron.

The role of the striatum in the control of movements and in the processing of cortical information has received much attention in the recent years. We set out a simple biophysical model for the medium-spiny neuron (msn), the most abundant cell in striatum. This neuron receives two main kinds of inputs, namely, cortical excitatory inputs and dopaminergic inputs coming from the substantia nigra pars compacta. The msn axon impinges directly onto the globus pallidus and onto the substantia nigra pars reticulata neurons and onto striatal neurons through recurrent branches of the axon. The msn is characterized by spiny dendritic trees with a high density of spines (1 to 4 spines/microns) and the probable existence of dendritic spikes. The model predicts that the neuron can integrate excitable inputs in a linear or a nonlinear mode. In the nonlinear mode, the neuron allows the detection of simultaneous (or almost simultaneous) synaptic inputs; it facilitates either a slowing down or a speeding up of the information transfer between the synaptic input location and the soma and is sensitive to inhibiton-excitation pairing. Conversely, in the linear integrative mode, the somatic voltage is determined by a weighted summation of the synaptic inputs. Several geometrical, electrical, or temporal factors can control the switch between these behaviors: the density of excitable dendritic elements, the dendritic radius, the resistance of the spine stem, the membrane resistance, the time between excitations, and the distance between synaptic sites. Finally, the signification of this behavior is discussed in connection with the putative role of dopamine and with the striatal net organization.

Immunohistochemical study of the catecholaminergic innervation of the spinal cord of the rat using specific antibodies against dopamine and noradrenaline.

We have assessed the relative contributions of dopaminergic and noradrenergic descending systems to the catecholaminergic innervation of the rat spinal cord. Fibres and terminals were labelled with their own neurotransmitter by using specific antibodies raised against dopamine (DA) and noradrenaline (NA) respectively. For this purpose, immunohistochemistry according to the peroxidase anti-peroxidase technique was performed in different experimental conditions. Two group of rats received intracisternal 6-hydroxy-dopamine (6-OHDA) injections either with or without benzatropine pretreatment. Animals of a third group were not pretreated at all. While 6-OHDA induced a complete disappearance of spinal NA-like immunoreactivity (NA-LI), except for scarce residual fibres in the thoracic intermedio-lateral cell column, DA-like immunoreactivity (DA-LI) was unaffected by the lesion. This strongly suggests that the antisera used specifically labelled NA-containing and DA-containing fibres respectively. Spinal DA-LI and NA-LI innervations differed markedly in their topographical distributions and in the morphology of the corresponding fibres. DA-LI innervation was restricted to laminae I, III and IV and to the intermediate zone, especially the autonomic areas. In the ventral horn, it was sparse and more visible after acidification of the fixation solution. NA-LI innervation was much more widely spread. In addition, the organization of NA-LI fibres suggests that the innervation of the whole dorsal horn comes from a group of fibres travelling, at least partially, in the superficial dorsal horn.

Rostrocaudal localization of cardiovascular responses induced by intrathecal administration of apomorphine in conscious, freely moving rats.

In conscious freely moving rats, administration of apomorphine (179 nmol), a dopamine receptor agonist, into the intrathecal (i.t.) space decreased mean aortic blood pressure (MBP) and heart rate (HR). Both the magnitude and the time of appearance of the response varied according to the spinal level of administration. The largest and immediately appearing effect was observed after the injection at the upper thoracic site, whereas the magnitude of the responses was smaller with an immediate or slightly delayed (0.5-1.5 min) onset at lower thoracic and midcervical levels. More caudal responses appeared to be due to spreading of the drug along the spinal axis (onset in 1-2 min after administration). Behavioral responses (stereotyped movements) were observed within 2-3 min after administration and were nearly the same whatever the site of administration. These results corroborate, as do those provided by i.t. injections of tritiated apomorphine, the spinal origin of cardiovascular effects of i.t. apomorphine. Furthermore, spinal transection at the T5-T7 level did not change the magnitude and duration of decreases in MBP and HR elicited by i.t. apomorphine injected at the T2-T4 level. Moreover, this procedure enhanced responses to i.t. administration at the T9-T10 level. In conclusion, these results favor the existence of a spinal site of action for the cardiovascular effects of apomorphine. Furthermore, they indicate that spinal transection is accompanied by development of a hypersensitive phenomenon (of a mechanism to be determined).

Evidence for the existence of L-dopa- and dopamine-immunoreactive nerve cell bodies in the caudal part of the dorsal motor nucleus of the vagus nerve.

The precise neurochemical nature of tyrosine hydroxylase-immunoreactive neurons lying in the caudal part of the dorsal motor nucleus of the vagus nerve of the rat has been identified by immunohistochemistry of the catecholamines themselves. This region corresponds precisely to the area where tyrosine hydroxylase has been previously shown to be colocalized with choline acetyltransferase. Adjacent serial cryostat sections from the medulla oblongata and from the cervical spinal cord were treated either for choline acetyltransferase immunohistochemistry, aromatic L-amino acid decarboxylase and tyrosine hydroxylase immunolabelling or for tyrosine hydroxylase, dopamine, noradrenaline and L-dihydroxyphenylalanine (DOPA) immunostaining. The procedure involved the peroxidase-antiperoxidase method and an intensified diaminobenzidine reaction with imidazole. While no noradrenaline-positive cells were detectable in the dorsal motor vagal nucleus, tyrosine hydroxylase-, dopamine- and DOPA-immunoreactive perikarya were seen in the medial half of this nucleus, caudally the obex level. These results led us to conclude that these tyrosine hydroxylase-positive cells were effectively of dopaminergic nature and therefore that dopamine is a neurotransmitter contained in some neurons of the dorsal motor vagal nucleus. In the light of previous data showing colocalization of tyrosine hydroxylase and choline acetyltransferase in neurons of this portion of the nucleus, colocalization of dopamine with acetylcholine appears most likely. This might shed some light on the physiological consequences of dopamine action at target parasympathetic organs, such as the gastrointestinal tract.

Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurones of the vagal motor nucleus in the adult rat.

Catecholaminergic nerve cell bodies have been recently identified in the rat spinal cord. They lie in the rostral cervical segments and at the lumbosacral junction. Among them, many are located in parasympathetic areas. This finding led us to investigate the interactions between these catecholaminergic neurones and the cholinergic ones. To address this question, we performed sequential immunocytochemical detection of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in the same sections. We could then identify the co-expression of both TH and ChAT-like immunoreactivities (LI) in some perikarya of the cervical spinal cord and medulla oblongata. Such cells are located in the caudal extension of the dorsal motor nucleus of the vagus nerve (DMNX) as well as in the caudal part of the medullary DMNX itself. Such a co-expression of TH-LI and ChAT-LI could not be found in the lumbosacral region, another parasympathetic territory where cell bodies displaying TH-LI were intermingled with those containing ChAT-LI. This is one of the first demonstrations of the co-existence of catecholaminergic and cholinergic phenotypes in some neurones of the adult mammalian nervous system. These observations also support the presence of catecholaminergic efferents within the vagus nerve.

[The spinal dopaminergic system]. / Le système dopaminergique spinal.

A new dopaminergic innervation has been described quite recently: the dopaminergic spinal cord system. In this review are presented the different steps which lead to the individualization of this dopaminergic system and the reported results actually available concerning its probable anatomical organization. Finally, most of the data which illustrate the possible functions of this system are discussed. Interestingly, its participation in the transmission of nociceptive signals and the control of cardiovascular patterns appear now well established. Such functional implications give new information on the possible targets of central dopaminergic agonists and antagonists, particularly at the cardiovascular level.

Immunohistochemical study of catecholaminergic cell bodies in the rat spinal cord.

Immunohistochemistry of three specific synthesizing catecholamine enzymes was used in the rat spinal cord to determine precisely the distribution of catecholaminergic perikarya and the nature of the neurotransmitter they contain. Single and double labeling experiments were performed on cryostat sections from perfused rats. The peroxidase anti-peroxidase (PAP) and the indirect fluorescence techniques were used for labeling spinal catecholaminergic somata and separated into two completely different populations. The first is located in the upper cervical cord and includes three apparently distinct groups: a lateral cluster, of probably a noradrenergic nature, and two central subgroups where noradrenergic and dopaminergic neurons are intermingled. It is likely that these cervical cells represent caudal extensions of the medullary catecholaminergic cell groups. In the remaining cord, only tyrosine hydroxylase immunoreactive cell bodies have been found. Accordingly, this second population is probably dopaminergic. It is present almost exclusively in the first sacral segments, where it is located in the commissural (mostly lateral) grey matter and in the marginal dorsal horn.

Changes of the striatal 3H-spiperone binding 3-6 weeks after nigrostriatal denervation and after two years.

A complete unilateral lesion of the nigrostriatal pathway by 6-hydroxydopamine injection in the substantia nigra induced a drastic increase in striatal dopaminergic binding sites labelled by 3H-spiperone, 30 days after the lesion. This increase (75% over controls) was time restricted: it was only 39% and 34% over control values at respectively 25 and 35 days after the lesion. Furthermore, 45 days after the destruction of the substantia nigra, the density of labelled sites returned close to the homolateral control values, but remained higher than the contralateral ones, according to the right-left difference found in control animals. Quite later (2 years after the lesion), there was a decrease in the density of labelled sites as compared to the respective homolateral control levels. However, such binding sites tend to remain higher in the striatum of the lesioned side than in the striatum of the intact one, although such a difference was not statistically significant, being very close to the right-left asymmetry observed in control animals. Contrary to our previous results with 3H-Haloperidol, the apparent dissociation constant did not vary significantly, whatever the considered delay after the lesion. These results are discussed in the light of previous results obtained by others and by us.

Immunohistochemical demonstration of catecholaminergic cell bodies in the spinal cord of the rat. Preliminary note.

In order to elucidate the anatomy of the spinal dopaminergic system, an immunohistochemical study using a tyrosine-hydroxylase (TH) antibody was undertaken in the rat. Intracisternal 6-hydroxydopamine (6-OHDA) injections were administered to destroy most of the noradrenergic fibres that descend to the spinal cord while preserving the dopaminergic fibres. The density of the remaining TH-like immunoreactive fibres was relatively low at all levels of the spinal cord; the highest density was observed in layers III, IV and X. In addition, we report the first evidence for the existence of TH-like immunoreactive cell bodies at definite levels (especially sacral) of the spinal cord.

Cardiovascular effects in the rat of intrathecal injections of apomorphine at the thoracic spinal cord level.

Intrathecal (i.t.) administration of apomorphine at the upper thoracic level lowered blood pressure and heart rate in awake rats. This decrease was dose-dependent and competitively antagonized by haloperidol (i.v. and i.t.) or domperidone (i.t.) but not by domperidone (i.v.). Furthermore, these effects of apomorphine were not affected by alpha- and beta-blocking drugs (i.t.). The results suggest a spinal site, at least in part, for the cardiovascular effect of apomorphine.

Dissociate destruction of noradrenaline and dopamine descending projections in the thoracic spinal cord of the rat.

Intracisternal administration of 6-hydroxydopamine to male Wistar rats produced a near complete depletion of noradrenaline levels, as measured by a radioenzymatic assay in micropunches sampled from the dorsal, lateral and ventral horns of the thoracic spinal cord. This drastic effect was reversed by pretreatment with desipramine, a pharmacological inhibitor of noradrenergic neuron uptake. Surprisingly, dopamine content was not significantly reduced. The question as to whether such a lack of concomitant dopamine decrease might be inherent to the dopamine assay itself could be answered by the results obtained with both pharmacological (reserpine) treatments and interference determinations in the dopamine assay. The relative potency of 6-hydroxydopamine to destroy noradrenergic and dopaminergic neurons might account for their differential behavior. Conversely, a large midbrain section performed by knife cut could decrease both dopamine and DOPAC (one of its major acid metabolites) in the thoracic lateral horn and partly in the ventral one. The noradrenaline content was not reduced. Results are discussed in light of recently reported data on dopaminergic descending projections to the spinal cord. The lesion procedures presented here seem to provide valuable tools to dissociate noradrenergic from dopaminergic spinal projections, which is necessary for further anatomical and functional studies on these systems.

Supersensitivity time course of dopamine antagonist binding after nigrostriatal denervation: evidence for early and drastic changes in the rat corpus striatum.

The sensitivity of the [3H]haloperidol binding technique can be greatly increased by focusing tissue sampling on striatal regions where dopaminergic innervation is the richest. Such sampling is provided from pooled microdiscs punched out of 8 serial 500 micrometer thick sections of the rat brain. With these conditions, the density of receptor sites (Bmax) was found to be twice that of the whole striatum, without modification of the apparent dissociation constant (Kd) and of the Hill's slope. Such a procedure applied to rats with complete 6-hydroxydopamine-induced unilateral nigrostriatal lesions showed a moderate decrease in Bmax in the lesioned side up to 6 days after surgery, whereafter the value of Bmax increased progressively up to the thirtieth day, being then 160% over the control value. Conversely, the apparent Kd decreased significantly from the second to the sixth day postsurgery in the lesioned side, and then increased moderately up to the tenth day and drastically from the twenty-first to the thirtieth day. No change was observed in the corresponding intact side. The modifications appeared chronologically compatible with those corresponding to the behavioral denervation supersensitivity, evidencing drastic binding changes as compared with the whole striatum. The unexpected variations in Kd observed were well correlated with those in Bmax, suggesting that the new available binding sites might be of lower affinity. In the light of all these results, a hypothetical model is proposed.