Biomedical subjects

B Torres

Publications and source records attributed to B Torres.

At least 37 records · Page 2Linked to original sources

PubMed2000

Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), a putative P2Y(1) receptor antagonist, blocks signaling at a site distal to the receptor in Madin-Darby canine kidney-D(1) cells.

Substantial evidence documents the potential importance of P2Y receptor subtypes in the regulation of cellular responses, but few selective antagonists exist for these receptors. In the current study, we assessed the use of pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate (PPADS) as a putative P2Y(1) receptor-selective blocker in Madin-Darby canine kidney (MDCK-D(1)) cells. We found that the key action of PPADS in MDCK-D(1) cells was blockade of signaling at a postreceptor site. PPADS blocked UTP (P2Y(2))-stimulated accumulation of cAMP [which is dependent on arachidonic acid (AA) metabolism by cyclooxygenase] but not that by 2-methyl thio-adenosine triphosphate (2MeSATP; which is independent of cyclooxygenase and has been attributed to P2Y(1) and P2Y(11) receptors). By contrast, PPADS inhibited AA release mediated by both 2MeSATP and UTP. PPADS displayed uncompetitive antagonism in blockade of AA release in response to 2MeSATP. PPADS also inhibited AA release stimulated by various nucleotides, phenylephrine, and bradykinin, implying that the effect does not involve the inhibition of a specific receptor. Because PPADS also inhibited ionomycin-, thapsigargin-, and phorbol-12-myristate-13-acetate-promoted AA release, it appears to act at a site distal to an increase in intracellular Ca(2+) transients or PKC activation. Inhibition of melittin-stimulated AA release by PPADS suggested that the target of PPADS action may either be a phospholipase A(2) (PLA(2)) or a site distal to PLA(2), but PPADS did not inhibit Ca(2+)-dependent PLA(2) activity in MDCK-D(1) cell homogenate. The data indicate that PPADS blocks AA release in response to multiple compounds and suggest caution in the use of this compound for distinguishing P2Y receptor subtypes.

Adenosine Triphosphate↗

PubMed1999

Tectotectal connectivity in goldfish.

The vertebrate optic tectum is a functionally coupled bilateral structure which plays a major role in the generation of motor commands for orienting responses. However, the characteristics of the tectotectal connectivity are unknown in fish, and have been reported only to a limited extent in other vertebrates. The purpose of the present study was to determine the anatomical basis underlying the functional coupling between tecta in goldfish, and to identify both similarities and differences to those features reported in other vertebrate species. The present experiments used the bidirectional tracer biotinylated dextran amine to map the distribution of labeled cells and synaptic boutons in the contralateral tectum following injections into identified tectal sites. Fibers that interconnect both tecta coursed through the tectal commissure. The cells of origin of these fibers, the tectotectal cells, and their synaptic endings were located in the deep layers, mainly in the strata periventricular and griseum central, respectively. Corresponding sites throughout the two tecta were interconnected in a symmetrical point-to-point fashion. The tectal commissure was composed of at least two distinct bundles of axons, which differed in their dorsoventral location, fiber diameter, and projection targets. The dorsal axons were tectotectal axons, they were thinner in diameter and profusely branched, and gave off en passant and terminal boutons in the deep layers of the contralateral tectum. The ventral axons were thicker in diameter, and formed the contralateral tectofugal-descending tract. Such fibers had few axon collaterals and boutons in the contralateral tectum. Boutons adjacent to retrogradely labeled tectotectal cells were very scarce. The data are discussed in terms of the coupling between tecta generating the motor commands required for orienting movements.

Animals↗

PubMed1999

A method for measuring eye movements using Hall-effect devices.

A system for precise recording of eye position and movements in laboratory animals, by means of Hall-effect devices, is described. The system, useful in neurophysiological and neurobehavioral studies, allows the analysis of saccadic eye movements, optokinetic- and vestibular-induced nystagmus, slow tracking movements, eye vergences, and so forth. This small, light-weight, and inexpensive system uses a set of Hall-effect devices and associated electronics to sense variations in the position of high-power magnets fixed in the eye sclera or in scleral contact lenses. The output of the Hall-effect devices is amplified by operational amplifiers, collected through an A/D converter, and analyzed in a PC computer by specific software.

Animals↗

PubMed1998

IFNgamma inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21WAF1/CIP1.

Glioblastoma is a highly aggressive form of brain cancer characterized by uncontrolled cell growth resulting from a loss of cell cycle regulation. In this study we determined the antiproliferative effects of interferon gamma (IFNgamma) on the glioblastoma cell lines T98G, SNB-19 and U-373, focusing on the ability of IFNgamma to increase levels of p21WAF1/CIP1, an important negative regulator of cell cycle events. IFNgamma was found to inhibit the growth of all cell lines, with inhibition ranging from 82.2% to 45.4%. Flow cytometry analysis showed that IFNgamma treatment caused a cell cycle delay in the G1 or S phases. The strength of this delay varied, correlating with the degree by which IFNgamma inhibited proliferation of each cell line. IFNgamma treatment increased the production of the cyclin dependent kinase inhibitor (CKI) p21WAF1/ CIP1 in all cell lines, the level and kinetics of production of which correlated with the degree and stage of inhibition of cellular proliferation. Further, immunoprecipitation of p21WAF1/CIP1 in complexes of p21WAF1/CIP1/cyclin-dependent kinase 2 (cdk2)/cyclin showed that the amount of p21WAF1/CIP1 in the complexes and the inhibition of cdk2-cyclin kinase activity correlated with the level of p21WAF1/CIP1 produced in the cells by IFNgamma. These results show that IFNgamma has significant antiproliferative effects on the glioblastoma cell lines and suggest that p21WAF1/CIP1 plays a role in mediating these effects.

CDC2-CDC28 Kinases↗

PubMed1998

Influence of the tectal zone on the distribution of synaptic boutons in the brainstem of goldfish.

This study investigated whether the topographic differences in the functional properties of the tectal motor map of goldfish are related to particular patterns of connections with downstream structures. With this aim, the distribution of synaptic boutons in the mesencephalic and rhombencephalic structures was studied after discrete injections of the tracer biotinylated dextran amine were placed at separate sites along the tectal anteroposterior axis. Irrespective of the location of the injection site, the boutons were more abundant in the mesencephalon than in the rhombencephalon, and they were located chiefly ipsilaterally all throughout the brainstem. In the mesencephalon, the boutons were found in its ventrolateral reticular formation and, to a lesser extent, in the nucleus of the medial longitudinal fasciculus, the oculomotor and isthmi nuclei, and the torus semicircularis. In the mesencephalic reticular formation, the bouton location was distributed topographically with respect to the injection site. Terminals were also observed in the nucleus of the medial longitudinal fasciculus after injections into anteromedial or middle tectal zones. In the oculomotor nucleus, boutons were present exclusively in the case of the anteromedial injection. In the rhombencephalon, most boutons were found in the superior reticular formation, and their number decreased in the medial and inferior reticular formations. A topographic distribution could be observed within the superior reticular formation, although its density was attenuated compared with that observed in the mesencephalic reticular formation. The domains of synaptic endings on the ipsilateral side were different from those on the contralateral side: The ipsilateral synaptic endings were located more medially. Finally, a few boutons were also found in the vestibulocerebellar area on either the ipsilateral or the contralateral side, depending on the injection site. From these data, the authors conclude that, in goldfish, irrespective of the tectal injection site, the endings are in similar nuclei in the brainstem; however, the distribution of synaptic boutons within such nuclei can be related to the functional properties of each tectal zone.

Anatomy, Artistic↗

PubMed1998

Tail and eye movements evoked by electrical microstimulation of the optic tectum in goldfish.

This work studies the tail and eye co-ordinated movements evoked by the focal electrical stimulation of the tectum in goldfish. The aim of the study is to understand better those tectal sites and mechanisms that either remain functionally unaltered or are adaptively modified across vertebrates. Stimulation was applied in various tectal zones, and the characteristics of evoked tail and eye movements were examined as a function of the stimulation site over tectal surface and the stimulus parameters. Two types of response were electrically evoked: the former turned the body and the eyes contraversively towards the source of natural stimulus; the second produced initial ipsiversive turning of the body and eyes, followed by several tail beats. Evoking one or other response depended on both the site and parameters of stimulation, and responses were interpreted as orienting- and escape-like, respectively. Depending on the stimulation site, four different zones in the tectum were distinguished: in the medial zone the stimulus elicited eye and tail movements whose size increased with the distance to the rostral pole. The stimulation of the antero-medial zone evoked contraversive or ipsiversive eye saccades but tail movements were similar, irrespective of eye movements. Stimulation within the extreme antero-medial zone evoked convergent eye movements, and tail displacements turning the body either ipsiversively or contraversively. Stimulation of the posterior zone often evoked complex tail movements and pure horizontal eye saccades. Both orienting- and escape-like responses were also dependent on the stimulus parameters. The relationships between stimulus parameters and tail- and eye-orienting movement characteristics suggest that the velocity and duration might be encoded in different aspects of the tectal activity. Current strength also modified the number of tail beats that appeared during escape-like response. In conclusion, the present data suggest the involvement of the optic tectum not only in orienting but also in escape responses and that movements of eye and tail mediating such responses depend on the tectal active locus together with its level of activity.

Animals↗

PubMed1998

10-Oximeguanacone, the first nitrogenated acetogenin derivative found to be a potent inhibitor of mitochondrial complex I.

A new 10-keto bis-tetrahydrofuran acetogenin, guanacone (1), has been isolated from a cytotoxic extract of Annona aff. spraguei seeds. The 10-oximeguanacone derivative 1f is the first bioactive nitrogenated acetogenin found to be a very potent inhibitor of complex I. In addition, a SAR study of guanacone analogues is reported based on the titration of the NADH oxidase and NADH:ubiquinone oxidoreductase activities.

Animals↗

PubMed1997

Tectal codification of eye movements in goldfish studied by electrical microstimulation. f.

This work compares the tectal codification of eye movements in goldfish with those reported for other vertebrate groups. Focal electrical stimulation was applied in various tectal zones and the characteristics of evoked eye movements were examined as a function of (i) the position of the stimulation over the tectal surface, (ii) the initial position of the eyes and (iii) the parameters (pulse rate, current strength, duration) of the stimulus. In a large medial zone, stimulation within the intermediate and deep layers of the tectum evoked contraversive saccades of both eyes, whose direction and amplitude were roughly congruent with the retinotopic representation of the visual world within overlying layers. These saccades were minimally influenced by the initial position of the eye in the orbit. The topographical arrangement of evoked saccades and body movements suggests that this tectal zone triggers orienting responses in a similar way to those described in other vertebrates. Stimulations applied within the caudal tectum also evoked contraversive saccades, but in disagreement with the overlying retinotopic map--the vertical component was absent. Taken together with electrically evoked body movements reported in free-swimming fish, these saccades could reveal that this zone is involved in escape responses. When stimulations were applied within the anteromedial zone of the tectum, contraversive movements of both eyes appeared much more dependent on initial eye position. Saccades elicited from this area displayed characteristics of "goal-directed saccades" which were similar to those described in the cat. The generation of goal-directed movements from the anteromedial zone suggests that this portion of the goldfish optic tectum has a different intrinsic organization or is connected with the brainstem saccade generator in a different fashion than the medial zone. Finally, stimulation of the extreme anteromedial zone evoked convergent eye movements. These movements and those reported in free-swimming fish following electrical stimulation of this tectal area suggest that this zone could be involved in feeding responses. The relationships between the parameters of electrical stimulation and the characteristics of elicited saccades suggest that the stimulated location within the tectum determines a constant direction in the evoked saccade, whereas the amount and duration of tectal activity, as mimicked by changes in stimulus parameters, together with the tectal locus, determine the velocity and amplitude of the evoked saccade.

Animals↗

PubMed1997

A hospital/school science fair mentoring program for middle school students.

The Massachusetts General Hospital (MGH) and the James P. Timilty Middle School established a partnership to enhance science education, promote faculty development, and improve the health status and academic performance of all Timilty students. This article describes one of the Partnership's Science Connection programs, the Science Fair Mentoring Program, designed to enhance middle school science education, inform urban early adolescents about professions in the health field, inspire them to pursue postsecondary study in the health sciences, and prepare them for rigorous academic work in high school. In this program, hospital-based clinical and research staff mentor young adolescent students. The authors describe the planning, implementation, and evaluation of the Science Fair Mentoring Program as an innovative learning experience.

Adolescent↗

PubMed1996

Telencephalic ablation in goldfish impairs performance in a 'spatial constancy' problem but not in a cued one.

This work was aimed to study if goldfish telencephalon is involved differentially in spatial and cue learning. With this purpose, animals were assigned to two learning conditions, 'spatial constancy' and 'directly cued', and their performance was recorded before and after ablation of the telencephalon. During the presurgical acquisition period, animals of both groups learned to solve the task with accuracy, and reached the goal in transfer tests, even though they were released from new starting positions and the response requirements were changed. Ablation impaired selectively the solution of the spatial constancy problem, but had no significant effects on the cued condition. However, with additional training, performance of the ablated animals in the spatial constancy condition improved to control levels. The above data suggest that fishes can implement multiple spatial learning strategies which have different neural substrata. These results are discussed in relation to the possible nature of the representation underlying each task.

Animals↗

PubMed1996

Spatial learning and memory deficits after telencephalic ablation in goldfish trained in place and turn maze procedures.

The present work investigated whether the fish telencephalon is involved in spatial learning based on place strategies in a manner similar to mammalian hippocampus. Goldfish were trained in a 4-arm maze in a room with relevant spatial cues. Sham and to-be-ablated subjects were trained in each of 4 experimental procedures designed as follows: place, turn, place-turn, and control. After acquisition, complete ablations of both telencephalic hemispheres for the experimental groups were carried out. The results showed that ablation exclusively impaired performance in animals using place strategies; in these, accuracy fell to chance level during both postsurgery retraining and reversal periods. In the other groups, ablation of the telencephalon did not induce any significant deficit. These results suggest that the fish telencephalon plays a crucial role in complex place learning.

Animals↗

PubMed1996

Enhanced levels of wild-type versus mutant thyroid hormone receptor beta 1 messenger RNA in fibroblasts from heterozygotes of kindred S with thyroid hormone resistance.

Thyroid hormone resistance syndromes, which result from heterozygous mutations in the beta 1 thyroid hormone receptor gene, are sometimes associated with adult short stature, but more frequently with delayed bone age (BA). Primary fibroblasts from young children with both delayed BA and short stature from a kindred A have been reported to overexpress the mutant allele. However, in fibroblasts from affected members of two different kindreds with thyroid hormone resistance, S and Mf, there were equal levels of mutant and wild-type beta 1 mRNA. We investigated the ontogeny of differential allelic expression using competitive reverse transcription with PCR (RT-PCR) to measure relative mRNA levels for beta 1 and S receptor in very young affected children of kindred S. Total RNA was prepared from fibroblasts of two patients (ages 3-0.5/12 and 1-4/12 years) with delayed BA but normal growth curves. Using PCR amplimers that create an Mlu-1 site in wild-type but not mutant cDNA products from the competitive RT, we quantitated mRNA levels. Normal beta 1 mRNA was present at nearly twice the level of the mutant mRNA in cells from these patients. Relative expression of the c-erbA beta alleles thus appeared to be increased during this period of somatic growth. The relative overexpression of the normal allele potentially counteracted the potent dominant negative effect of the S receptor during early childhood ameliorating a deleterious effect on linear growth.

Age Determination by Skeleton↗

PubMed1995

Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase.

We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 micrograms/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol.min-1.mg-1, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol.min-1.mg-1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol.min-1.mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis.

Amino Acid Oxidoreductases↗

PubMed1995

Distribution of neurons projecting to the trochlear nucleus in goldfish (Carassius auratus).

The distribution of neurons projecting to the trochlear nucleus of goldfish (Carassius auratus) was studied by the electrophoretic injection of horseradish peroxidase into the nucleus. The location of the injection site was electrophysiologically determined by the antidromic field potential elicited from the trochlear nucleus after the electrical stimulation of its nerve. Retrogradely labeled neurons were observed in the following structures: (1) anterior, tangential and descending nuclei of the octaval column--afferents to these nuclei were mainly ipsilateral for the former and exclusively contralateral for the other two; (2) cerebellum; (3) rhombencephalic reticular formation, near the abducens nucleus; and (4) nucleus of the medial longitudinal fasciculus. In addition, a few stained neurons were scattered in the nucleus of the posterior commissure and in nucleus pretectalis superficialis pars magnocellularis. These results are compared with the afferent sources to trochlear nucleus in mammals and with the set of structures projecting to the oculomotor and abducens nuclei in goldfish. It is suggested that the neuronal pathways involved in the compensatory vestibular and optokinetic reflexes, as well as in the saccadic system, are similarly organized in vertebrates but that the relative importance of some of the components of these pathways varies.

Abducens Nerve↗

PubMed1993

Competitive polymerase chain reaction quantitation of c-erbA beta 1, c-erbA alpha 1, and c-erbA alpha 2 messenger ribonucleic acid levels in normal, heterozygous, and homozygous fibroblasts of kindred S with thyroid hormone resistance.

Mutations in the T3-binding domain of the thyroid hormone receptor gene c-erbA beta result in dominant negative proteins and thyroid hormone resistance syndromes. Variable clinical manifestations of resistance to thyroid hormones have been reported, including short stature and neuropsychological abnormalities. The molecular bases for heterogeneity of phenotype among and within kindreds have not been fully elucidated. Recent investigations have considered differential expression of mutant and wild-type beta 1-receptor alleles and the regulation thereof as a mechanism to explain differential sensitivity to thyroid hormones. We used reverse transcription-competitive polymerase chain reaction (PCR) to measure c-erbA beta 1, c-erbA alpha 1, and c-erbA alpha 2 mRNAs in skin fibroblasts cultured from normal subjects, heterozygotes, and a severely affected homozygous mutant of kindred S. The homozygous mutant of kindred S had severe growth and mental retardation. After reverse transcription with primers specific for each of the c-erbA mRNAs, first strand cDNAs were amplified by PCR using subtype-specific amplimers. Primer design allowed simultaneous detection of wild-type and mutant messages in heterozygous fibroblasts and showed an approximately 1:1 ratio of these mRNAs in three patients. Inclusion of competitive standard cDNAs of known concentration in the PCR reactions allowed quantitation of the absolute levels of the beta 1-, alpha 1-, and alpha 2 mRNAs by comparison of products on ethidium bromide-stained agarose gels. These studies showed no effect of the presence of the mutant beta 1-allele, as fibroblast RNA from normal subjects, heterozygotes, and the homozygote gave values of 56-184, 2.8-12, and 23-40 attomol/5 micrograms total RNA for beta 1-, alpha 1-, and alpha 2 mRNAs, respectively. We conclude that these sensitive methods allow the detection of molecular species present at levels as low as 10 molecules/cell, and that this potent dominant negative receptor does not disrupt c-erbA expression at the level of mRNA. The neuropsychological sequelae of the kindred S mutation are not due to relative overexpression of the mutant allele.

Alleles↗

PubMed1992

Afferents to the oculomotor nucleus in the goldfish (Carassius auratus) as revealed by retrograde labeling with horseradish peroxidase.

The goal of this work was to compare the distribution and morphology of neurons projecting to the oculomotor nucleus in goldfish with those previously described in other vertebrate groups. Afferent neurons were revealed by retrograde labeling with horseradish peroxidase. The tracer was electrophoretically injected into the oculomotor nucleus. The location of the injection site was determined by the antidromic field potential elicited in the oculomotor nucleus by electrical stimulation of the oculomotor nerve. Labeled axons whose trajectories could be reconstructed were restricted to the medial longitudinal fasciculus. In order of quantitative importance, the afferent areas to the oculomotor nucleus were: (1) the ipsilateral anterior nucleus and the contralateral tangential and descending nuclei of the octaval column. Furthermore, a few labeled cells were found dorsomedially to the caudal pole of the unlabeled anterior octaval nucleus; (2) the contralateral abducens nucleus. The labeled internuclear neurons were arranged in two groups within and 500 microns behind the caudal subdivision of the abducens nucleus; (3) a few labeled cells were observed in the rhombencephalic reticular formation near the abducens nucleus, most of which were contralateral to the injection site. Specifically, stained cells were found in the caudal pole of the superior reticular nucleus, throughout the medial reticular nucleus and in the rostral area of the inferior reticular nucleus; (4) eurydendroid cells of the cerebellum, located close to the contralateral eminentia granularis pars lateralis, were also labeled; and (5) a small and primarily ipsilateral group of labeled cells was located at the mesencephalic nucleus of the medial longitudinal fasciculus. The similarity in the structures projecting to the oculomotor nucleus in goldfish to those in other vertebrates suggests that the neural network involved in the oculomotor system is quite conservative throughout phylogeny. Nevertheless, in goldfish these projections appeared with some specific peculiarities, such as the cerebellar and mesencephalic afferents to the oculomotor nucleus.

Afferent Pathways↗

PubMed1992

A morphological study of abducens nucleus motoneurons and internuclear neurons in the goldfish (Carassius auratus).

The location and distribution of abducens (ABD) nucleus motoneurons (Mn) and internuclear neurons (Int) were determined in the goldfish (Carassius auratus) by means of horseradish peroxidase and fluorochrome retrograde labeling. ABD Mn were labeled following tracer injection into the ipsilateral lateral rectus muscle. These Mn were arranged in two ventrolateral clusters along the rostro-caudal axis of the posterior brainstem. Both groups of neurons showed a similar number of cells, and their axons ran ventrally to their respective nerve roots. ABD Int were labeled following the injection of the tracer into the contralateral oculomotor nucleus. They also formed two distinct groups in the rostro-caudal axis. The rostral group of Int formed a dorso-lateral cap around the caudal motoneuronal pool, with little if any intermingling. The caudal group of Int was located at the same position in the dorso-ventral and medio-lateral axis as the rostral group, but 500 microns behind it. Both groups of ABD Int had a similar number of neurons. Int axons ascended dorso-medially, then crossed the midline through the internal arcuate fibers, and entered the contralateral medial longitudinal fasciculus. The soma diameters of both ABD Mn and Int were not significantly different. The relative location of both types of neurons is discussed.

Animals↗

PubMed1992

Effects of diazepam and D-amphetamine on rhythmic pattern of eye movements in goldfish.

The effects of diazepam and D-amphetamine on spontaneous, vestibular- and visually-induced eye movements were studied in the restrained goldfish. These animals showed a stereotyped pattern of spontaneous eye movements that scanned the visual field in the horizontal plane. Fourier analysis of these eye movements revealed a rhythmic pattern of about 5 cycles per minute, whose frequency increased to double and decreased to half following treatment with diazepam and D-amphetamine, respectively. However, the fast and slow components of vestibulo-ocular and optokinetic reflexes, as well as peculiar blink-like movements, remained unchanged at the same drug doses. These results suggest the presence of a central pattern generator for spontaneous eye movements in the goldfish, whose oscillation frequency is pharmacologically modifiable.

Animals↗