Ultrastructure of Motor Nerve Terminals in the Anterior Third of Wistar Rat Tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4 degrees C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi`s apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech. 72:464-470, 2009. (C) 2009 Wiley-Liss. Inc.

Physiological roles and regulation of mammalian sulfate transporters

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients; in cells and is the fourth most abundant anion in human plasma (300 muM). Sulfate is the major sulfur source in many organisms, and because it is a hydrophilic anion that cannot passively cross the lipid bilayer of cell membranes, all cells require a mechanism for sulfate influx and efflux to ensure an optimal supply of sulfate in the body. The class of proteins involved in moving sulfate into or out of cells is called sulfate transporters. To date, numerous sulfate transporters have been identified in tissues and cells from many origins. These include the renal sulfate transporters NaSi-1 and sat-1, the ubiquitously expressed diastrophic dysplasia sulfate transporter DTDST, the intestinal sulfate transporter DRA that is linked to congenital chloride diarrhea, and the erythrocyte anion exchanger AE1. These transporters have only been isolated in the last 10-15 years, and their physiological roles and contributions to body sulfate homeostasis are just now beginning to be determined. This review focuses on the structural and functional properties of mammalian sulfate transporters and highlights some of regulatory mechanisms that control their expression in vivo, under normal physiological and pathophysiological states.

The ribonucleic acid content of some mammalian erythrocytes

RNA was determined in red blood cells of man and other mammals. Our report is based on 41 determinations. Red blood cells of rat showed the highest values in comparison with the blood cells of guinea pig, rabbit, horse and sheep which showed the lowest values, and man with intermediate ones. The method used was a combination of Schimidt and Thanhauser and Schneider extractions with the final reactions of pentose with the orcinol reagent colorimetrically measured.

Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophylicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.

Desenvolvimento de imunossensor impedimétrico para detecção do corante disperso Red 1

Pós-graduação em Química - IQ

Rapid screening of potential autophagic inductor agents using mammalian cell lines

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.

Synaptic circuitry of ganglion cells in the mammalian retina

Before signals of the visual environment are transferred to higher brain areas via the optic nerve, they are processed and filtered in parallel pathways within the retina. In the past a plethora of functionally distinct ganglion cell types responding to certain aspects of the environment, such as direction of movement, contrast and colour have been described. Aim of this thesis was the anatomical investigation of the selectivity in retinal circuits underlying this diversity. For this purpose, mouse and macaque retinae were analysed. OFF-ganglion cells in the mouse retina received their excitatory drive unselectively from all bipolar cell types stratifying within the area of their dendritic trees. Only the input to direction-selective C6 ganglion cells and bistratified D2 ganglion cells appeared to be weighted. In primates the highly specialised midget-system forms a 1:1 connection from red- and green-sensitive cones onto midget bipolar- and ganglion cells, building the substrate for red/green colour vision. Here it was demonstrated that blue-sensitive (S-) cones also contact OFF-midget bipolars and are, thus, potential candidates to transfer blue-OFF signals to M1 intrinsically photosensitive ganglion cells (ipRGCs). M1 cells received glycinergic input from A8 amacrine cells and express GABAA receptors containing subunit alpha 3. M2 cells, in contrast, received less inhibitory input.

STUDIES ON THE BIOLOGICAL ACTIVITY OF MACROMOLECULES MICROINJECTED INTO MAMMALIAN SOMATIC CELLS

Red Blood cell mediated and glass needle mediated microinjection technology was used to introduce macromolecules into mammalian somatic cells. The biological activities of DNA synthesis inducing factor(s) (Chapter 1), mitotic factor(s) (Chapter 2), and DNA coding for ovalbumin and thymidine kinase (Chapter 3) were studied following injection into mammalian somatic cells.^ Chapter 1. A cell undergoing DNA replication (S phase) contains a factor(s) that induces DNA synthesis prematurely in a G(,1) nucleus when an S phase cell is fused to a G(,1) cell. An assay for the active factor(s) was developed in which a mixture of s phase extract loaded red blood cells (RBC) and synchronous G(,1) HeLa cells was centrifuged onto Concanavalin A (Con A) treated coverslips and fused by PEG. This technique is called "Centrifusion". The synchronous G(,1) HeLa cells injected with S phase extract initiated DNA synthesis earlier than the control G(,1) cells mock injected with RBC loaded with buffer.^ Chapter 2. It has been demonstrated that fusion between a mitotic and an interphase cell usually leads to breakdown of the interphase nucleus, followed by condensation of the interphase chromatin into discrete chromosomes, a process termed premature chromosome condensation. I wanted to develop an assay for the mitotic factor(s) that induces premature chromosome condensation. Experiments were performed utilizing glass needle mediated microinjection of HeLa cell mitotic extract into interphase somatic mammalian cells in an attempt to induce premature chromosome condensation. However, I was not able to induce premature chromosome condensation in the interphase cells, probably because of an inability to introduce sufficient mitotic factor(s) into the cells.^ Chapter 3. A recombinant plasmid containing the chicken ovalbumin gene and three copies of the Herpes thymidine Kinase gene (pOV12-TK) was introduced into mouse LMTK('-) cell nuclei using glass needle mediated gene transfer resulting in LMTK('+) clones that were selected for in HAT medium. Restriction enzyme analysis of the high molecular weight DNA from 6 HAT medium survivor cell clones revealed the presence of one or at best only a few copies of the 12kb ovalbumin gene per mouse genome. Further analysis showed the ovalbumin DNA was not rearranged and was associated with high molecular weight mouse cell DNA. Each of the analyzed cell clones produced ovalbumin demonstrating that the biological activity of the microinjected ovalbumin was retained. ^

Structural protein 4.1 is located in mammalian centrosomes

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.

Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian CellsV⃞

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds.

To explore the evolutionary dynamics of genes in the major histocompatibility complex (Mhc) in nonmammalian vertebrates, we have amplified complete sequences of the polymorphic second (beta1) and third (beta2) exons of class II beta chain genes of songbirds. The pattern of nucleotide substitution in the antigen-binding site of sequences cloned from three behaviorally and phylogenetically divergent songbirds [scrub jays Aphelocoma coerulescens), red-winged blackbirds (Agelaius phoeniceus), and house finches (Carpodacus mexicanus) reveals that class II B genes of songbirds are subject to the same types of diversifying forces as those observed at mammalian class II loci. By contrast, the tree of avian class II B genes reveals that orthologous relationships have not been retained as in placental mammals and that, unlike class II genes in mammals, genes in songbirds and chickens have had very recent common ancestors within their respective groups. Thus, whereas the selective forces diversifying class II B genes of birds are likely similar to those in mammals, their long-term evolutionary dynamics appear to be characterized by much higher rates of concerted evolution.

Phytochemical Markers Of Different Types Of Red Propolis.

Propolis is a resin that bees collect from different plant sources and use in the defense of the bee community. The intricate composition of propolis varies depending on plant sources from different geographic regions and many types have been reported. Red coloured propolis found in several states in Brazil and in other countries has known antimicrobial and antioxidant activity. Different analytical methods have been applied to studies regarding the chemical composition and plant origins of red propolis. In this study samples of red propolis from different regions have been characterised using direct infusion electrospray ionisation mass spectrometry (ESI(-)-MS) fingerprinting. Data from the fingerprints was extracted and analysed by multivariate analysis to group the samples according to their composition and marker compounds. Despite similar colour, the red coloured propolis samples were divided into three groups due to contrasting chemical composition, confirming the need to properly characterise the chemical composition of propolis.

Antibody Recognition Of Plasmodium Falciparum Infected Red Blood Cells By Symptomatic And Asymptomatic Individuals In The Brazilian Amazon.

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Understanding Red Blood Cell Parameters In The Context Of The Frailty Phenotype: Interpretations Of The Fibra (frailty In Brazilian Seniors) Study.

Frailty and anemia in the elderly appear to share a common pathophysiology associated with chronic inflammatory processes. This study uses an analytical, cross-sectional, population-based methodology to investigate the probable relationships between frailty, red blood cell parameters and inflammatory markers in 255 community-dwelling elders aged 65 years or older. The frailty phenotype was assessed by non-intentional weight loss, fatigue, low grip strength, low energy expenditure and reduced gait speed. Blood sample analyses were performed to determine hemoglobin level, hematocrit and reticulocyte count, as well as the inflammatory variables IL-6, IL-1ra and hsCRP. In the first multivariate analysis (model I), considering only the erythroid parameters, Hb concentration was a significant variable for both general frailty status and weight loss: a 1.0g/dL drop in serum Hb concentration represented a 2.02-fold increase (CI 1.12-3.63) in an individual's chance of being frail. In the second analysis (model II), which also included inflammatory cytokine levels, hsCRP was independently selected as a significant variable. Each additional year of age represented a 1.21-fold increase in the chance of being frail, and each 1-unit increase in serum hsCRP represented a 3.64-fold increase in the chance of having the frailty phenotype. In model II reticulocyte counts were associated with weight loss and reduced metabolic expenditure criteria. Our findings suggest that reduced Hb concentration, reduced RetAbs count and elevated serum hsCRP levels should be considered components of frailty, which in turn is correlated with sarcopenia, as evidenced by weight loss.