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Biomedical subjects

E Barbosa

Publications and source records attributed to E Barbosa.

At least 37 records · Page 2Linked to original sources
PubMed1993

Retinoic acid induces changes in the rhombencephalic neural crest cells migration and extracellular matrix composition in chick embryos.

Chick embryos at 9-10 stages (Hamburger and Hamilton: J Morphol 88:49-82, 1951) have been treated with all-trans retinoid acid (RA) (0.5 microgram, 1.5 micrograms, and 2.5 micrograms) to determine the pattern and mechanism of RA-induced effects on early cephalic development. We found that while 0.5 microgram RA did not produce any significant dysmorphogenesis, 2.5 micrograms RA elicited wide malformation of both cephalic and trunk regions. However, 1.5 micrograms RA produced selective and specific changes at the cephalic level, which consisted of morphological alterations, changes in neural crest cells (NCC) migration and extracellular matrix (ECM) composition. Morphological alterations included hypoplasia of the first three branchial arches, swelling of either anterior cardinal veins or dorsal aortae, and atrophy of branchial arch arteries. Concurrently NCC did not migrate away, remaining clustered on the dorsal surface of the rhombencephalon, and in some cases they shifted into the neural tube cavity. Accordingly, the second branchial arch showed a reduction of the mesenchymal cellular population. The extracellular matrix in RA-injected embryos showed changes in glycosaminoglycans (GAGs) concentration as compared with controls, that is, an increase in the non-sulphated GAGs, stained with alcian blue 8GX at 2.5 pH, and a decrease in the sulphated GAGs stained with alcian blue 8GX at 1 pH. These quantitative changes reflected alterations in the pattern of distribution and composition of the GAGs within the cephalic ECM, which specifically consisted in an increase of the hyaluronic acid and a decrease of the chondroitin sulphate. Our findings indicate that RA is involved in abnormal cephalic development, suggesting that RA may effect neural crest cell migration via changes in the GAGs of the ECM.

Animals↗
PubMed1993

Chondroitin sulphate proteoglycan and embryonic brain enlargement in the chick.

Previous studies of the early development of the neural tube have shown the existence of an intraneural fluid, which causes a positive pressure inside this primordium, and seems to play a key role in the early development of the central nervous system. In the present study we investigated the composition and synthesis of this intraneural fluid. By using a sequential method, which includes fixation with glutaraldehyde plus cetylpyridinium chloride, opening the neural cavity after critical point drying and scanning electron microscopy analysis, we found a water-soluble extracellular matrix that filled up the brain vesicles of chick embryos at the earliest stages of the neural tube. An ultrastructural study of the neural epithelium during these stages revealed the existence of a secretion process in the neural cells toward the apical side, the future neural cavity. An immunocytochemical study to assess the nature of the secreted material has shown that the intraneural matrix contains chondroitin sulphate proteoglycan, which appeared homogeneously distributed throughout the neural cavity. Our findings demonstrate that the intraneural liquid is a fluid of complex composition and includes chondroitin sulphate proteoglycan as an osmotically active molecule. This suggests a morphogenetic role for the proteoglycan during early brain enlargement. The neural ectoderm is a polarized epithelium from early developmental stages and secretes the intraneural matrix.

Animals↗
PubMed1993

Morphological study by scanning electron microscopy of the lingual papillae in the common European bat (Pipistrellus pipistrellus).

There are many accounts of the tongues of mammals observed with scanning electron microscopy (SEM), but apparently only one article about the tongue of the Chiroptera. In the present study the tongue surface (after removing extracellular material) of the European common bat (Pipistrellus pipstrellus) was examined. The tongue is covered with papillae and has an elevation between its medial and posterior half. There are three types of papillae: filiform, fungiform and circumvallate, and the filiform can be classified as strictly filiform, conical and crown-like. The shapes and disposition of these papillae are related to function, which is principally to retain captured food during flight. At higher magnification the surface of the filiform papillae has many pores and microridges, which may serve for the production and distribution of mucus over the papillary surface.

Animals↗
PubMed1993

Refsum disease: a defect in the alpha-oxidation of phytanic acid in peroxisomes.

The oxidation of phytanic acid to pristanic acid was previously demonstrated to be deficient in monolayer cultures of skin fibroblasts (Herndon et al. 1969. J. Clin. Invest. 48: 1017-1032). However, identification of subcellular organelle with deficient enzyme activity has not been established. To define the subcellular organelle with deficient enzyme activity in the catabolism of phytanic acid, we measured the oxidation of [1-14C] phytanic acid to 14CO2 and pristanic acid in different subcellular organelles isolated from cultured skin fibroblasts from control and Refsum patients. The rates of oxidation of phytanic acid in peroxisomes, mitochondria, and endoplasmic reticulum were 37.1 +/- 2.65, 1.9 +/- 0.3, and 0.4 +/- 0.07 pmol/h per mg protein, respectively, from control fibroblasts. The phytanic acid oxidation activity in mitochondria (2.04 +/- 0.7 pmol/h per mg protein) and endoplasmic reticulum (0.43 +/- 0.2 pmol/h per mg protein) from Refsum fibroblasts was similar to control fibroblasts. However, phytanic acid oxidation in peroxisomes from Refsum fibroblasts was not detected at all the protein concentrations tested. On the other hand, the peroxisomes from Refsum fibroblasts had normal rates of activation and oxidation of palmitic and lignoceric acids, suggesting that the peroxisomes isolated from Refsum fibroblasts were metabolically active. The phytanoyl-CoA ligase, the first enzyme in the alpha-oxidation pathway, had activity similar to that in peroxisomes from control (9.86 +/- 0.09 nmol/h per mg protein) and Refsum (10.25 +/- 0.31 nmol/h per mg protein) fibroblasts. The data described here clearly demonstrate that pathognomonic accumulation of phytanic acid in patients with Refsum disease is due to the deficient activity of peroxisomal alpha-oxidation enzyme system.

Cells, Cultured↗
PubMed1990

Patterns of epithelial cell death during early development of the human inner ear.

A light microscopic study of cell death in a developmental series of otic primordia from 23 human embryos (Carnegie stages 9 to 14) was completed. Degenerated cells were noted predominantly in the placode (stages 9 and 10), cup (stages 11 and 12), and otocyst (stages 13 and 14). A systematic camera lucida study of the appearance and topography of degenerating epithelial cells showed four different areas of cell death in the otic primordia that related to 1) invagination and detachment of the otic anlage, 2) early histogenesis of the statoacoustic ganglion, and 3) development of the endolymphatic duct. The possible role of cell death in the morphogenesis of the inner ear related to morphogenetic movements is discussed.

Cell Survival↗
PubMed1988

Characterization of a cloned cDNA encoding rabbit myelin P2 protein.

Myelin P2 is a 14,800-Da cytosolic protein found in rabbit sciatic nerves. It belongs to a family of fatty acid binding proteins and shows a 72% amino acid sequence similarity to aP2/422, the adipocyte lipid binding protein, a 58% sequence similarity to rat heart fatty acid binding protein, and a 40% sequence similarity to cellular retinoic acid binding protein. In order to isolate cDNA clones representing P2, a cDNA library was constructed from poly(A+) RNA isolated from sciatic nerves of 10-day-old rabbit pups. By use of a mixed synthetic oligonucleotide probe based on the rabbit P2 amino sequence, 12 cDNA clones were selected from about 25,000 recombinants. Four of these were further characterized. They contained an open reading frame, which when translated, agreed at 128 out of 131 residues with the known rabbit P2 amino acid sequence. These cDNAs recognize a 1.9-kilobase mRNA present in sciatic nerve, spinal cord, and brain, but not present in liver or heart. The levels of P2 mRNA parallel myelin formation in sciatic nerve and spinal cord with maximal amounts being detected at about 15 postnatal days. This initial study will allow characterization of the P2 gene and its regulation, as well as further studies into the role of P2, the first metabolically active myelin-specific protein to be characterized at the genetic level.

Amino Acid Sequence↗
PubMed1988

Bombesin and other growth factors activate cell proliferation in chick embryo otic vesicles in culture.

The ability of the mitogenic peptide bombesin and other growth factors to trigger and support early development of the inner ear was studied on chick embryo otocysts in culture. The normal pattern of development was preserved in cultured otic vesicles in the presence of 20% fetal calf serum in the medium. Differentiation proceeded from stage 18 to 22 during the first 24 h and further to stage 24 in 48 h. Estimates of cell number and mitotic rates revealed a distinct period of proliferative growth which was maximum at the 24 h period of incubation. This was coincident with a high rate of DNA synthesis as measured by the acid-precipitable incorporation of [3H]thymidine. Development could be arrested by deprivation of serum during 24h. It could then be reactivated by readmission of serum to proceed with the normal pattern of morphological differentiation and cell proliferation. Bombesin (100 nM) was able to reactivate development in growth-arrested vesicles. Its effect was dose-dependent, saturable and potentiated by insulin (5 micrograms ml-1) which was ineffective if used alone. When associated with insulin, bombesin carried differentiation to stage 21 and stimulated mitotic activity above the level of serum as judged from estimates of cell number and [3H]thymidine uptake. EGF and PDGF were also effective in reinitiating development although their potency was smaller than bombesin. The reactivation by serum or bombesin was blocked by amiloride. The results show that (1) the otic vesicle can provide a useful model for studying the mechanisms that control proliferative growth and differentiation during normal development and (2) bombesin and other growth factors are able to activate growth in embryonic developing tissues.

Animals↗
PubMed1987

Spatial segregation of mRNA encoding myelin-specific proteins.

The cellular and subcellular distributions of mRNAs encoding three myelin-specific proteins--myelin basic protein (MBP), proteolipid protein (PLP), and Po protein--were studied in tissue sections of developing rat nervous systems by in situ hybridization. The developmental appearance of these mRNAs closely paralleled the appearance of the proteins they encode as determined by immunocytochemistry. mRNA encoding the extrinsic membrane protein, MBP, was concentrated around oligodendrocyte and Schwann cell nuclei during initial stages of myelination; as myelination proceeded, MBP mRNA became distributed diffusely over myelinated fibers. In contrast, mRNAs encoding the intrinsic membrane proteins, PLP and Po, remained concentrated around oligodendrocyte (PLP) and Schwann cell (Po) nuclei at all stages of myelination. These results establish that myelinating cells spatially segregate certain myelin-specific mRNAs. The presence of MBP mRNA within the cytoplasmic domains of myelin internodes indicates that protein sorting during myelination involves transportation of mRNA to specific subcellular sites.

Aging↗
PubMed1987

Polarization and density of Na-pumps in the inner ear of the chick embryo during early stages of development.

The otic vesicle consists of a pseudostratified epithelium with some features of transporting epithelia. The present work questions whether Na-pumps are polarized in this epithelium and what is the relation between the location or density of pumps and development. This was done by measuring the binding of [3H]ouabain to isolated otic vesicles in developmental stages 16 to 22. The results show the presence of specific ouabain-binding sites located in the inward-facing membrane of the otic vesicle epithelium. Binding was saturable at increasing concentrations of ouabain and was highly sensitive to the external K+ concentration with half-maximal inhibition below 0.5 mM, indicating that the binding sites and Na-pump sites are identical. A transient stage-dependent increase in the density of Na-pumps during the period that precedes growth was observed. Evidence is given against this being directly related to an increased net fluid transport rate despite the fact that pump sites were always polarized throughout these stages. The conclusions are that (1) the otic vesicle epithelium is a polarized structure with the possibility of vectorial transport of solutes and water and (2) the increased number of pumps may operate as a regulatory mechanism during normal proliferative growth.

Animals↗
PubMed1986

Electrical properties of the otic vesicle epithelium in the chick embryo.

The electrophysiological properties of the epithelium of the otic vesicle were studied in the chick embryo using conventional microelectrode techniques. A preparation is described that allows continuous recording of transmural potential and resistance during changes in the composition of the bathing fluid. Vesicles in stages 18 to 22 showed a spontaneous transmural potential (ET) that ranged from 2 to 6 mV, inner positive. This electrical potential difference was abolished after 2 h incubation in K+-free strophantidin (10(-4) M) and it increased by about twofold immediately after addition of the cation ionophore Amphotericin B (250 microM) to the bath. The specific resistance of the wall (RT) was about 80 omega cm2 between stages 18 and 22 indicating a low-resistance, noncellular, permeation pathway for current flow. The short-circuit current, calculated from ET and RT was about 50 X 10(-6) A cm-2 throughout this period. This corresponds to a net flux of 187 X 10(-8) mol cm-2 h-1 of a single cation pumped towards the towards the vesicular cavity. Diffusion potentials (salt gradients and single-ion substitutions) showed a selectivity ratio PK:PNa:PCl = 1:0.9:0.7, which is that of a weakly charged aqueous pathway. Measurements of vesicular volume and surface area showed an increase by a factor of ten in the size of the vesicle with maximal rates of change in volume of 5 microliter cm-2 h-1. The electrical properties reported here for the epithelium of the otic vesicle resemble very much those of 'leaky' epithelia which are known to transport ions and water at a very high rate.

Amphotericin B↗
PubMed1980

Inverted terminal repetition in vaccinia virus DNA encodes early mRNAs.

Vaccinia virus DNA contains a long inverted terminal repetition of MW approximately 6.8 x 10(6). A fragment of MW 6.3 X 10(6) from this repetition has been cloned in coliphage lambda and used to isolate RNA from virus-infected cells. Electron microscopy indicates that early RNAs are transcribed from the repeated sequence and cell-free translation shows that the RNAs code for polypeptides.

Bacteriophage lambda↗
PubMed1980

Expression of the vaccinia virus genome: analysis and mapping of mRNAs encoded within the inverted terminal repetition.

We have investigated the organization of transcriptional units within a 9000 bp segment of the terminally repeated region of the DNA genome of vaccinia virus, which uses its own enzyme system to synthesize mRNA within the cytoplasm of infected cells. RNA splicing, which has been demonstrated for DNA viruses that replicate within the nucleus of infected cells, does not appear to be involved in the formation of these first vaccinia virus mRNAs to be examined. Three immediate early mRNAs, approximately 1050, 600 and 1100 nucleotides long, were mapped between 3.21 and 4.24, 6.54 and 7.16, and 7.20 and 8.23 kb from the end of the genome, respectively. The direction of transcription was toward the end of the genome for the two larger mRNAs and in the opposite direction for the smallest one. Additional minor RNAs, which were larger in size, were mapped between and to the same DNA strand as the mRNAs of 1050 and 1100 nucleotides. No evidence for interrupted genes was obtained by nuclease S1 analysis after hybridization of RNA to labeled DNA. In addition, the 5' ends of the mRNAs, which were specifically labeled by in vitro capping, hybridized to DNA adjacent to the body of the message.

DNA, Recombinant↗
PubMed1978

mRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Purification and physical properties.

An S-adenosyl-L-methionine:mRNA(nucleoside-2'-)-methyltransferase, one of at least three activities required for the 5'-terminal modification of mRNA, has been purified from vaccinia virus particles. Employing brome mosaic virus RNA ending in m7G(5')pppG- as substrate, a simple DEAE-cellulose filter assay measuring the incorporation of methyl groups from S-adenosyl[methyl-3H]methionine to position 2' of the penultimate nucleoside was devised. Starting from disrupted vaccinia virus cores, a 350-fold enzyme purification was achieved by successive chromatography on columns of DEAE-cellulose, CM-Sephadex, and APP-agarose. Analysis of the isolated enzyme by sodium dodecyl sulfate-polyacrylamide discontinuous gel electrophoresis revealed a single polypeptide with a molecular weight of 38,000. Similar molecular weights were obtained by sucrose gradient centrifugation and gel filtration of the native methyltransferase. The isoelectric point of the purified enzyme occurs at pH 8.4.

Methyltransferases↗
PubMed1978

mRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Characteristics and substrate specificity.

An mRNA(nucleoside-2'-)-methyltransferase purified from vaccinia virus was shown to methylate the penultimate nucleoside of RNA ending in m7G(5')pppN-. By contrast, RNAs ending in pN-, ppN-, or even G(5')pppN- are not methyl acceptors. This specificity indicates that 2'-O-methylation is the final step in the formation of the m7G(5')pppNm- cap structure. Both adenosine and guanosine are methylated, in accordance with the presence of these nucleosides in the penultimate position of vaccinia virus mRNAs. Studies with homopolyribonucleotides containing m7G(5')pppN ends indicated that poly(A) and poly(I) were the best methyl acceptors while significant but much less activity was obtained with poly(G), poly(U), and poly(C). Simple dinucleotides of the type m7G(5')pppN, however, are poor substrates and do not compete with capped RNA. Additional studies indicate that the methyltransferase has a pH optimum of 7.5, does not require divalent cations, is inhibited by S-adenosylhomocysteine, has a Km of 2.0 micrometer for S-adenosylmethionine, a Km of approximately 5 nM for brome mosaic virus RNA, and kinetics consistent with a random bireactant mechanism.

Kinetics↗
PubMed1978

Visualization of an inverted terminal repetition in vaccinia virus DNA.

An inverted terminal repetition was observed in DNA molecules extracted from vaccinia virus. The repeated sequence was visualized by (i) nicking the hairpin loops present of the ends of vaccinia virus DNA, (ii) separating the strands of DNA by alkali denaturation, (iii) allowing the single strands to self-anneal, and (iv) examining the DNA with an electron microscope. Single-stranded circular molecules, each of which contained a duplex projection (3.54 +/- 0.12 micron) representing the terminal repetition, readily formed. Similar size projections were also seen in heteroduplex structures formed by crosshybridization of the separated strands of the two terminal HindIII restriction fragments. Based on contour length measurements and the electrophoretic mobility of the isolated inverted terminal repetition, a molecular weight of approximately 6.9 X 10(6), equivalent to about 10,500 nucleotide base pairs, was estimated. Evidence was obtained from DNA-RNA hybridization studies that the terminal repetition is transcribed.

Base Sequence↗
PubMed1975

Nascent DNA from phytohemagglutinin-stimulated human lymphocytes.

DNA synthesis was studied in human peripheral blood lymphocytes that had been stimulated with phytohemagglutinin. DNA pulse-labeled with [3H]thymidine was fractionated by sucrose gradient centrifugation or by chromatography on hydroxylapatite columns. Nascent DNA was identified as a single-stranded species that sedimented at 4-5S in neutral sucrose gradients and appeared to be precursor to chromosomal DNA in pulse-chase experiments. At least two-thirds of the nascent DNA was released as single strands from high molecular weight DNA without employing a denaturation step. It is concluded that synthesis of DNA by phytohemagglutinin-stimulated lymphocytes involves a low molecular weight, single-stranded, short-lived intermediate similar to that described for other eukaryotic cells.

Cell Separation↗