Biomedical subjects

L R Britto

Publications and source records attributed to L R Britto.

At least 19 recordsLinked to original sources

PubMed2006

The effect of exposure to irrigant solutions on apical dentin biofilms in vitro.

This study assessed the effectiveness of different concentrations of sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) (Vista Dental Products, Racine, WI), and BioPure MTAD (Dentsply Endodontics-Tulsa Dental, Tulsa, OK). Intracanal contents were collected from 10 patients diagnosed with chronic apical periodontitis. The samples were cultured on hemisections of root apices to generate a polymicrobial biofilm. Each biofilm was separately immersed in 6% NaOCl, 3% NaOCl, 1% NaOCl, 2% CHX, 1% NaOCl followed by BioPure MTAD, and sterile phosphate buffered solution (PBS). SEM analysis showed 6% NaOCl and 3% NaOCl were capable of disrupting and removing the biofilm; 1% NaOCl and 1% NaOCl followed by MTAD were capable of disrupting the biofilm, but not eliminating bacteria; 2% CHX was not capable of disrupting the biofilm. Viable bacteria could not be cultured from specimens exposed to 6% NaOCl, 2 % CHX, or 1% NaOCl followed by BioPure MTAD. These results indicate that 6% NaOCl was the only irrigant capable of both rendering bacteria nonviable and physically removing the biofilm.

Biofilms↗

PubMed2001

Expression of endo-oligopeptidase A in the rat central nervous system: a non-radioactive in situ hybridization study.

Endo-oligopeptidase A (EOPA, formerly EC 3.4.22.19), a thiol-activated oligopeptidase, is able to degrade both bradykinin and neurotensin, and also to convert enkephalin-containing peptides into enkephalins. The expression of this enzyme was studied in the rat brain by in situ hybridization using non-radiotopic probes. The distribution of EOPA transcripts included many regions of the rat central nervous system, with higher expression in some regions, such as the hippocampus, cerebellum, and basal nucleus of Meynert. The marked EOPA expression in these areas could be anticipated, since they are rich in neuropeptides that are known to be EOPA substrates in vitro. The data characterize a widespread occurrence of EOPA in the rat brain and reinforce the suggestion of a critical role for EOPA in peptide processing.

Animals↗

PubMed2001

Differential localization of acetylcholinesterase in relation to pre- and postsynaptic nicotinic receptors in the chick brain.

Immunofluorescence and confocal microscopy were combined to study the distribution of acetylcholinesterase in relation to the localization of the beta2 subunit of the nicotinic acetylcholine receptors in the chick brain. In several areas where the beta2 subunit is recognizably part of presynaptic receptors, the localization of acetylcholinesterase appeared not to overlap the localization of beta2. On the other hand, acetylcholinesterase and the beta2 subunit exhibited a strictly matching localization in areas where postsynaptic nicotinic receptors are known to be present. These data may represent a morphological substrate for possible differential actions of acetylcholinesterase at presynaptic and postsynaptic nicotinic sites.

Acetylcholinesterase↗

PubMed2001

Frequent occurrence of nicotinic acetylcholine receptors in GABAergic neurons of the chick visual system.

Double-labeling immunohistochemical methods were used to investigate the occurrence of the alpha8 and alpha5 nicotinic receptor subunits in presumptive GABAergic neurons of the chick nervous system. Nicotinic receptor immunoreactivity was often found in cells exhibiting GABA-like immunoreactivity, especially in the visual system. The alpha8 subunit appeared to be present in presumptive GABAergic cells of the ventral lateral geniculate nucleus, nucleus of the basal optic root of the accessory optic system, and the optic tectum, among several other structures. The alpha5 subunit was also found in GABA-positive neurons, as observed in the lentiform nucleus of the mesencephalon and other pretectal nuclei. The numbers of alpha8- and alpha5-positive neurons that were also GABA-positive represented high percentages of the total number of neurons containing nicotinic receptor labeling in several brain areas, which indicates that most of the alpha8 and alpha5 nicotinic receptor subunits are present in GABAergic cells. Taken together with data from other studies, our results indicate an important role of the nicotinic acetylcholine receptors in the functional organization of GABAergic circuits in the visual system.

Animals↗

PubMed2000

Expression of cholinergic system molecules during development of the chick nervous system.

There are suggestions of the participation of nicotinic acetylcholine receptors (nAChRs), the acetylcholine degradation enzyme, acetylcholinesterase (AChE), and the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), in the development of the nervous system. In this study, we aimed at comparing the development of some subunits of the nAChRs, AChE, and ChAT in the chick nervous system by standard immunohistochemical methods. The expression of all molecules investigated here appeared very early in ganglia (embryonic day 3.5-4), persisting into posthatching, except for ChAT, which is not detected after hatching in ganglia. A differential development was observed for nAChR subunits, with these receptors appearing around embryonic day 6 in some sites. The time-course of development of different nAChR subunits revealed several instances of transient expression (such as in the cerebellum), increasing expression (such as in the nucleus spiriformis lateralis), and diminishing expression into posthatching stages (such as in the oculomotor and throclear nuclei). Expression of AChE and ChAT also starts around embryonic day 6 in some structures and follows mainly increasing time-courses in the chick brain. The results of this study reveal a developmentally regulated expression of cholinergic system-related molecules in the chick nervous system and characterize differential time-courses of expression for nAChR subunits, AChE, and ChAT during development.

Acetylcholinesterase↗

PubMed2000

Late developmental expression of DARPP-32 in the chick optic tectum.

Immunohistochemical techniques reveal that the dopamine- and cAMP-regulated phosphoprotein DARPP-32 is detectable in neurons of the chick optic tectum starting on embryonic day 13. The expression levels then increase steadily from embryonic day 15 through the first posthatching day. After 15 days posthatching, expression of DARPP-32 reaches the adult pattern, with many labeled cells in tectal layers 11 and 12. These cells exhibit a bipolar shape, with long processes directed both to the deep and superficial layers. These results suggest that DARPP-32 is present in specific neuronal populations of the chick tectum and that this protein may not have a function in early ontogenetic processes.

Animals↗

PubMed2000

Effects of cholinergic deafferentation upon the expression of the alpha2 subunit of the nicotinic acetylcholine receptors in the chick lateral spiriform nucleus.

Lesion, immunohistochemical, and immunoblotting methods were used to evaluate the effects of cholinergic deafferentation upon the expression of the alpha2 subunit of the nicotinic acetylcholine receptors in the lateral spiriform nucleus (SpL) of the chick brain. The expression of the alpha2 subunit in the SpL showed biphasic changes after lesion of its cholinergic source (nucleus semilunaris), with an increase after 2 days postlesion and a decrease after 3-7 days. Our results could represent a correlate of the phenomena of nicotinic receptor up- and down-regulation, induced by removal of the cholinergic input.

Animals↗

PubMed2000

Retinal lesions induce differential changes in the expression of flip and flop isoforms of the glutamate receptor subunit GluR1 in the chick optic tectum.

A sensitive RNase protection assay was employed to determine the levels of mRNA encoding the GluR1 subunit flip and flop isoforms in the chick optic tectum and forebrain. We found that the flip GluR1 mRNA predominates in the forebrain, whereas the flop variant is more strongly expressed in the optic tectum. A temporal analysis of GluR1 variants in the embryonic and adult chick brain revealed that the flip isoform is more highly expressed at E12 than at P15-21, whereas mRNA levels of the flop isoform are higher at P15-21 than at E12. To study the effect of deafferentation on GluR1 expression, unilateral retinal lesions were performed. Two days later the mRNA levels of GluR1 flip and flop variants were decreased in the deafferented tectum, especially for the flop isoform. However, 7 days after the lesion, the mRNA levels of both GluR1 isoforms were increased, especially for the flip isoform. These results reveal an important control of the retinal input upon the expression of the different GluR1 isoforms. Furthermore, they indicate a differential spatial and temporal regulation of the flip and flop splice variants, suggesting the existence of a mechanism regulating differential splicing or possibly differential RNA stability.

Aging↗

PubMed2000

A comparative non-radioactive in situ hybridization and immunohistochemical study of the distribution of alpha7 and alpha8 subunits of the nicotinic acetylcholine receptors in visual areas of the chick brain.

The distribution of mRNA transcripts corresponding to the alpha7 and alpha8 subunits of the nicotinic acetylcholine receptors (nAChRs) was studied in selected structures of the chick visual system with non-radioactive in situ hybridization and immunohistochemical techniques. The results indicated that the alpha7 and alpha8 nAChR transcripts are widely distributed in the brain, exhibiting differential expression in some structures but also some degree of co-localization. The pattern of localization of alpha7 and alpha8 nAChR transcripts was highly correlated with immunohistochemical data, with very few instances of possible mismatches between the distribution of mRNAs and their corresponding proteins.

Animals↗

PubMed1999

Retinohypothalamic projections in the common marmoset (Callithrix jacchus): A study using cholera toxin subunit B.

Retinal projections in vertebrates reach the primary visual, accessory optic, and circadian timing structures. The central feature of the circadian timing system is the principal circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. The direct projections from the retina to the SCN are considered the entrainment pathway of the circadian timing system. In this study, unilateral intravitreal injections of cholera toxin subunit B were used to trace the retinal projections to the marmoset hypothalamus. The retinohypothalamic tract reaches the ventral suprachiasmatic nucleus bilaterally, as anticipated from previous studies. However, labeled fibers were found in several other hypothalamic regions, such as the medial and lateral preoptic areas, supraoptic nucleus, anterior and lateral hypothalamic areas, retrochiasmatic area, and subparaventricular zone. These results reveal new aspects of retinohypothalamic projection in primates and are discussed in terms of their implications for circadian as well as noncircadian control systems.

Animals↗

PubMed1999

Selective sensitivity of early postmitotic retinal cells to apoptosis induced by inhibition of protein synthesis.

In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.

Animals↗

PubMed1998

Expression of the AMPA-type glutamate receptor subunits in the chick optic tectum changes biphasically after retinal deafferentation.

Effects of retinal lesions on the expression of AMPA-type glutamate receptor (GluR) subunits in the chick optic tectum were evaluated with immunohistochemistry and immunoblotting. Expression of GluR1 and GluR2/3 subunits decreased in the deafferented tectum after 2 days and increased after 7 days postlesion. These results suggest biphasic effects of retinal lesions upon the expression of GluR subunits, possibly due to removal of the glutamatergic input from the retina.

Animals↗

PubMed1998

Distribution of the alpha7 nicotinic acetylcholine receptor subunit in the developing chick cerebellum.

Previous studies of the nicotinic acetylcholine receptor (nAChR) subunits in adult mammalian and avian brains have demonstrated a spatially restricted distribution of these subunits; little, however, is known about the nAChR subunit developmental distribution. The present study demonstrated a transient pattern of distribution of the neuronal nAChR subunit, alpha7, in the developing chick cerebellum by using immunohistochemical techniques. This transient distribution may suggest a critical period for the development of the cholinergic system in the cerebellum.

Animals↗

PubMed1997

Differential co-localization of nicotinic acetylcholine receptor subunits with calcium-binding proteins in retinal ganglion cells.

Immunohistochemistry was used to examine the co-occurrence of nicotinic acetylcholine receptor subunits with calcium-binding proteins in ganglion cells of the chick retina. The alpha3 subunit was rarely observed in ganglion cells containing calbindin, calretinin, or parvalbumin. On the other hand, the alpha8 subunit was more often co-localized with all calcium-binding proteins studied. These results may be related to the high calcium permeability of nicotinic receptors that contain the alpha8 subunit.

Animals↗

PubMed1997

Calbindin immunoreactivity delineates the circadian visual centers of the brain of the common marmoset (Callithrix jacchus).

The hypothalamic suprachiasmatic nucleus and the thalamic pregeniculate nucleus (which includes the intergeniculate leaflet) comprise the circadian visual system in the primate brain. In this study, we used intraocular injections of cholera toxin subunit B to identify those nuclei in the common marmoset brain, and demonstrated that calbindin D-28k immunoreactivity apparently labels most neurons in both the suprachiasmatic and pregeniculate nuclei. These data suggest that calbindin D-28k could represent a reliable neuronal marker for structures of the circadian visual system in marmosets and provide anatomical information on the primate equivalent of the rodent intergeniculate leaflet.

Animals↗

PubMed1997

Detection of calcium-binding proteins in venom and Duvernoy's glands of South American snakes and their secretions.

Calcium-binding proteins (CaBPs) have been described as involved in the stimulus-secretion coupling mechanisms in secretory glands. CaBPs were revealed with 45Ca, after electrophoresis in SDS-PAGE and transference to Zeta probe membranes, in Duvernoy's or venom gland homogenates from three families of South American snakes: Viperidae (Bothrops jararaca and Crotalus durissus terrificus); Elapidae (Micrurus corallinus), and Colubridae (Phylodrias patagoniensis and Oxyrhopus trigeminus). A band with an estimated molecular weight of 12 KDa was found in all glands studied. Bands with 17, 28, and 67 KDa were found in all glands, except in O. trigeminus Duvernoy's gland. A 18 KDa band was found in Viperidae and Elapidae venom glands, and a 88 KDa band was observed only in Viperidae venom gland homogenates. Some of these CaBPs were identified by Western blotting or by immunohistochemistry, as parvalbumin (12 KDa) and calbindin (28 KDa). When the secretion of these glands were analyzed, CaBPs were detected only in B. jararaca venom, with bands of 14, 35, 42, and 72 KDa. The profile of CaBPs was not modified at different phases of the secretory cycle of the glands, as well as after isoproterenol treatment.

Animals↗

PubMed1997

Control of venom production and secretion by sympathetic outflow in the snake Bothrops jararaca.

Many studies have examined the morphological and biochemical changes in the secretory epithelium of snake venom glands after a bite or milking. However, the mechanisms of venom production and secretion are not yet well understood. The present study was undertaken to evaluate the role of the sympathetic nervous system in the control of venom production and secretion. Venom glands were obtained from Bothrops jararaca (Viperidae) snakes, either unmilked previously or milked 4, 7 or 15 days before they were killed. Levels of tyrosine-hydroxylase-like immunoreactivity were higher in venom glands collected 4 days after milking, coinciding with the maximal synthetic activity of the secretory cells. The only catecholamine detected by high-performance liquid chromatography was noradrenaline, indicating the presence of noradrenergic fibres in these glands. In reserpine-treated milked snakes, no venom could be collected, and electron microscopic analysis showed narrow rough endoplasmic reticulum cisternae, instead of wide cisternae, and less well-developed Golgi apparatus compared with milked untreated snakes, indicating impairment of protein synthesis and secretion. The administration of isoprenaline or phenylephrine (beta- and alpha-adrenoceptor agonists, respectively) to reserpine-treated milked snakes promoted the widening of the rough endoplasmic reticulum and restored venom production, but only phenylephrine restored the development of the Golgi apparatus and the formation of many secretory vesicles. These results provide the first evidence that the sympathetic nervous system plays an important role in venom production and secretion in the venom glands of Bothrops jararaca. Understanding the importance of noradrenergic stimulation in venom production may provide new insights for research into the treatment of snakebites.

Animals↗

PubMed1997

Distribution of AMPA-type glutamate receptor subunits in the chick visual system.

Several glutamate receptor (GluR) subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex) were deeply anesthesized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GLuR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons.

Animals↗