RESUMO
Most, but not all cnidarian species in the class Hydrozoa have a life cycle in which a colonial, asexually reproducing hydroid phase alternates with a free-swimming, sexually reproducing medusa phase. They are not well known, in part because many of them are microscopic, at least in the medusa phase. Matching the two phases has previously required rearing of the organism from one phase to another, which has not often been possible. Here we show that DNA barcoding makes it possible to easily link life-cycle phases without the need for laboratory rearing. Hydrozoan medusae were collected by zooplankton tows in Newport Bay and the Pacific Ocean near Newport Beach, California, and hydroid colonies were collected from solid substrates in the same areas. Specimens were documented by videomicroscopy, preserved in ethanol, and sent to the Canadian Centre for DNA Barcoding at the University of Guelph, Ontario, Canada for sequencing of the COI DNA barcode. In the order Anthoathecata (athecate hydroids), DNA barcoding allowed for the discrimination between the medusae of eight putative species of Bougainvillia, and the hydroid stages were documented for two of these. The medusae of three putative species of Amphinema were identified, and the hydroid stages were identified for two of them. DNA barcodes were obtained from medusae of one species of Cladonema, one adult of the by-the wind Sailor, Velella velella, five putative species of Corymorpha with the matching hydroid phase for one; and Coryne eximia, Turritopsis dohrnii and Turritopsis nutricula with the corresponding hydroid phases. The actinula larvae and hydroid for the pink-hearted hydroid Ectopleura crocea were identified and linked by DNA barcoding. In the order Leptothecata (thecate hydroids) medusae were identified for Clytia elsaeoswaldae, Clytia gracilis and Clytia sp. 701 AC and matched with the hydroid phases for the latter two species. Medusae were matched with the hydroid phases for two species of Obelia (including O. dichotoma) and Eucheilota bakeri. Obelia geniculata was collected as a single hydroid. DNA barcodes were obtained for hydroids of Orthopyxis everta and three other species of Orthopyxis. One member of the family Solmarisidae, representing the order Narcomedusae, and one member (Liriope tetraphylla) of the order Trachymedusae were recognized as medusae. The results show the utility of DNA barcoding for matching life-cycle stages as well as for documenting the diversity of this class of organisms.
Assuntos
Biodiversidade , Código de Barras de DNA Taxonômico , Hidrozoários , Estágios do Ciclo de Vida/fisiologia , Zooplâncton , Animais , Hidrozoários/classificação , Hidrozoários/genética , Hidrozoários/crescimento & desenvolvimento , Microscopia de Vídeo , Oceano Pacífico , Zooplâncton/genética , Zooplâncton/crescimento & desenvolvimentoRESUMO
Despite the fact that Hydra has been studied for more than 200 years, we know surprisingly little about its life history. We show that Hydra vulgaris embryos hatch sporadically over a period ranging from a few days to nine months. We also report, for what seems to be the first time, the presence of Hydra in a vernal pool. Phylogenetic analysis and sexual crossing show that this Hydra is a member of the cosmopolitan Vulgaris clade and is not reproductively isolated from other members of the clade. Our findings lead us to hypothesize that Hydra evolved in an unstable freshwater habitat in which survival required that its life cycle include the use of a bet-hedging reproductive strategy and the formation of an embryo that is desiccation resistant and that can remain dormant for long periods of time.
Assuntos
Hydra , Animais , Filogenia , Reprodução , Estações do AnoRESUMO
Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals.
Assuntos
Adenosina Desaminase/fisiologia , Adenosina/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Animais , Diferenciação Celular/efeitos dos fármacos , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ecdisona/fisiologia , Corpo Adiposo/fisiologia , Hemócitos/enzimologia , Hemócitos/fisiologia , Larva/enzimologia , Metamorfose Biológica/efeitos dos fármacos , Mutação , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/fisiologia , Transdução de SinaisRESUMO
Adenosine receptors (AdoR) are members of the G protein-coupled receptor superfamily and mediate extracellular adenosine signaling, but the mechanism of adenosine signaling is still unclear. Here we report the first characterization of an insect AdoR, encoded by the Drosophila gene CG9753. Adenosine stimulation of Chinese hamster ovary cells carrying transiently expressed CG9753 led to a dose-dependent increase of intracellular cAMP and calcium, but untransfected controls showed no such response, showing that CG9753 encodes a functional AdoR. Endogenous CG9753 transcripts were detected in the brain, imaginal discs, ring gland and salivary glands of third-instar Drosophila larvae, and CG9753 overexpression in vivo caused lethality or severe developmental anomalies. These developmental defects were reduced by adenosine depletion, consistent with the proposed function of the CG9753 product as an AdoR. Overexpression of the G protein subunit Galpha(s) or of the catalytic subunit of protein kinase A (PKA) partially mimicked and enhanced the defects caused by ectopic expression of AdoR. Our results suggest that AdoR is an essential part of the adenosine signaling pathway and Drosophila offers a unique opportunity to use genetic analysis to study conserved aspects of the adenosine signaling pathway.
Assuntos
AMP Cíclico/fisiologia , Drosophila/metabolismo , Receptores Purinérgicos P1/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Drosophila/genética , Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hemolinfa , Injeções , Dados de Sequência Molecular , Fenótipo , Receptores Purinérgicos P1/genética , Alinhamento de SequênciaRESUMO
Mutations in apc which lead to activation of the Wnt signaling pathway are a hallmark of sporadic colon cancers but occur infrequently in colon cancers arising in patients with inflammatory bowel disease (IBD). There is evidence, however, that other components of the Wnt pathway may be altered in IBD-related colon cancer. In this study, we examined the expression the Wnt pathway components frizzled (Fz), the cell surface receptor, and disheveled (DVL), a family of cytoplasmic signal transduction molecules, in IBD and IBD-related colon cancer. Paraffin sections of normal and malignant colon tissues were obtained from patients with a history of ulcerative colitis and from controls with sporadic colon cancer. Tissue sections were stained with antibodies directed against Fz1/2 receptors and DVL1, DVL2 and DVL3 and antigen expression visualized by immunohistochemistry. Fz1/2 receptors were minimally expressed in normal IBD mucosa, were not expressed in IBD colon cancer, but exhibited strong expression in dysplastic tissues adjacent to the cancers. DVL1 was not expressed in IBD normal mucosa or normal mucosa from non-IBD patients, but was expressed in all cancers. DVL2 and DVL3 were expressed in all normal mucosa samples tested, and in sporadic colon cancer, but were not expressed in colon cancers arising in IBD patients. The characteristics of Fz and DVL expression in IBD tissues reported herein provides evidence of the importance of Wnt signaling in IBD and IBD-related colon cancer and, specifically, the significance of non-APC components of this pathway. Fz may serve as a marker for dyspasia in IBD patients and DVL1 is a potential therapeutic target for IBD-related colon cancer.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Doenças Inflamatórias Intestinais/genética , Proteína Wnt2/genética , Neoplasias do Colo/patologia , Receptores Frizzled/genética , Genes APC , Humanos , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Mucosa Intestinal/fisiologia , Mutação , Receptores Acoplados a Proteínas G/genética , Valores de ReferênciaRESUMO
Drosophila imaginal disc growth factor 2 (IDGF2) is a member of chitinase-like protein family (CLPs) able to induce the proliferation of imaginal disc cells in vitro. In this study we characterized physiological concentrations and expression of IDGF2 in vivo as well as its impact on the viability and transcriptional profile of Drosophila cells in vitro. We show that IDGF2 is independent of insulin and protects cells from death caused by serum deprivation, toxicity of xenobiotics or high concentrations of extracellular adenosine (Ado) and deoxyadenosine (dAdo). Transcriptional profiling suggested that such cytoprotection is connected with the induction of genes involved in energy metabolism, detoxification and innate immunity. We also show that IDGF2 is an abundant haemolymph component, which is further induced by injury in larval stages. The highest IDGF2 accumulation was found at garland and pericardial nephrocytes supporting its role in organismal defence and detoxification. Our findings provide evidence that IDGF2 is an important trophic factor promoting cellular and organismal survival.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/imunologia , Drosophila/metabolismo , Metabolismo Energético , Glicoproteínas/metabolismo , Imunidade Inata , Inativação Metabólica , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Hemolinfa/químicaRESUMO
Signaling complexes are essential for the modulation of excitability within restricted neuronal compartments. Adaptor proteins are the scaffold around which signaling complexes are organized. Here, we demonstrate that the Camguk (CMG)/CASK adaptor protein functionally modulates Drosophila Ether-á-go-go (EAG) potassium channels. Coexpression of CMG with EAG in Xenopus oocytes results in a more than twofold average increase in EAG whole-cell conductance. This effect depends on EAG-T787, the residue phosphorylated by calcium- and calmodulin-dependent protein kinase II (Wang et al., 2002). CMG coimmunoprecipitates with wild-type and EAG-T787A channels, indicating that T787, although necessary for the effect of CMG on EAG current, is not required for the formation of the EAG-CMG complex. Both CMG and phosphorylation of T787 increase the surface expression of EAG channels, and in COS-7 cells, EAG recruits CMG to the plasma membrane. The interaction of EAG with CMG requires a noncanonical Src homology 3-binding site beginning at position R1037 of the EAG sequence. Mutation of basic residues, but not neighboring prolines, prevents binding and prevents the increase in EAG conductance. Our findings demonstrate that membrane-associated guanylate kinase adaptor proteins can modulate ion channel function; in the case of CMG, this occurs via an increase in the surface expression and phosphorylation of the EAG channel.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas de Drosophila/fisiologia , Canais de Potássio Éter-A-Go-Go/fisiologia , Análise de Variância , Animais , Biotinilação/métodos , Western Blotting/métodos , Células COS , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Chlorocebus aethiops , Relação Dose-Resposta à Radiação , Drosophila , Proteínas de Drosophila/genética , Estimulação Elétrica/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Expressão Gênica/fisiologia , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Potenciais da Membrana/genética , Potenciais da Membrana/efeitos da radiação , Biologia Molecular/métodos , Dados de Sequência Molecular , Mutagênese/fisiologia , Mutação/fisiologia , Oócitos , Técnicas de Patch-Clamp/métodos , Fosforilação , Transporte Proteico/genética , Transporte Proteico/fisiologia , RNA Mensageiro/biossíntese , Ensaio Radioligante/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de Proteína/métodos , Transfecção/métodos , Xenopus , Domínios de Homologia de src/fisiologiaRESUMO
We report somatic mutations in three genes (CSNK1 epsilon, encoding the Ser/Thr kinase casein kinase I epsilon; DLG1, encoding a membrane-associated putative scaffolding protein; and EDD/hHYD, encoding a progestin induced putative ubiquitin-protein ligase) in mammary ductal carcinoma. These genes were suspected of playing a role in cancer because loss-of-function mutations in their Drosophila homologues cause excess tissue growth. Using DNA from 82 laser-microdissected tumor samples, followed by microsatellite analysis, denaturing HPLC and direct sequencing, we found multiple somatic point mutations in all three genes, and these mutations showed significant association with loss of heterozygosity of closely linked polymorphic microsatellite markers. For CSNK1 epsilon and DLG1, most of the mutations affected highly conserved residues, some were found repetitively in different patients, and no synonymous mutations were found, indicating that the observed mutations were selected in tumors and may be functionally significant. Immunohistochemical reactivity of each protein was reduced in poorly differentiated tumors, and there was a positive association between altered protein reactivity, loss of heterozygosity, and somatic mutations. There was a statistically significant association of hDlg staining with p53 and Ki67 reactivity, whereas CSK1 epsilon and EDD/hHYD staining levels were associated with progesterone receptor status. The results provide strong indications for a role of all three genes in mammary ductal carcinoma. They also justify additional studies of the functional significance of the changes, as well as a search for additional changes in these and other genes identified from studies on model systems.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Genes Supressores de Tumor , Mutação , Proteínas Quinases/genética , Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Estudos de Casos e Controles , Caseína Quinases , DNA de Neoplasias/genética , Proteína 1 Homóloga a Discs-Large , Feminino , Humanos , Perda de Heterozigosidade , Proteínas de Membrana , Dados de Sequência MolecularRESUMO
Many Drosophila genes exist as members of multigene families and within each family the members can be functionally redundant, making it difficult to identify them by classical mutagenesis techniques based on phenotypic screening. We have addressed this problem in a genetic analysis of a novel family of six adenosine deaminase-related growth factors (ADGFs). We used ends-in targeting to introduce mutations into five of the six ADGF genes, taking advantage of the fact that five of the family members are encoded by a three-gene cluster and a two-gene cluster. We used two targeting constructs to introduce loss-of-function mutations into all five genes, as well as to isolate different combinations of multiple mutations, independent of phenotypic consequences. The results show that (1) it is possible to use ends-in targeting to disrupt gene clusters; (2) gene conversion, which is usually considered a complication in gene targeting, can be used to help recover different mutant combinations in a single screening procedure; (3) the reduction of duplication to a single copy by induction of a double-strand break is better explained by the single-strand annealing mechanism than by simple crossing over between repeats; and (4) loss of function of the most abundantly expressed family member (ADGF-A) leads to disintegration of the fat body and the development of melanotic tumors in mutant larvae.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Conversão Gênica/fisiologia , Família Multigênica , Animais , Proteínas de Drosophila/fisiologia , Feminino , MasculinoRESUMO
Adenosine deaminases catalyze the deamination of adenosine and deoxyadenosine into their respective inosine nucleosides. Recent sequencing of the genomes of several model organisms and human reveal that Metazoa usually have more than one adenosine deaminase gene. A deficiency in the gene encoding the major enzyme is lethal in mouse and Drosophila and leads to severe combined deficiency (SCID) in human. In these organisms, enzyme deficiency causes increased adenosine/deoxyadenosine concentration in body fluids and some organs. Elevated levels of adenosine and deoxyadenosine are toxic to certain mammalian and insect cells, and it was shown for human and mouse that it is a primary cause of pathophysiological effects. Data suggest that the major role of adenosine deaminases in various taxa is the protection of tissues against increased levels of adenosine and deoxyadenosine. This review also discusses potential roles of adenosine deaminases in Drosophila metamorphosis and the employment of a Drosophila model to study the cell-specific toxicity of elevated nucleoside levels.
Assuntos
Adenosina Desaminase/metabolismo , Desoxiadenosinas/metabolismo , Insetos/fisiologia , Adenosina Desaminase/genética , Sequência de Aminoácidos , Animais , Humanos , Insetos/genética , Camundongos , Dados de Sequência MolecularRESUMO
We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.
Assuntos
Caseína Quinase 1 épsilon/genética , Caseína Quinase 1 épsilon/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila/crescimento & desenvolvimento , Drosophila/genética , Alelos , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Caseína Quinase 1 épsilon/química , Proliferação de Células , Drosophila/embriologia , Proteínas de Drosophila/química , Feminino , Humanos , Larva/genética , Dados de Sequência Molecular , Mutação , Fenótipo , Transdução de SinaisRESUMO
Stem cells are quickly coming into focus of much biomedical research eventually aiming at the therapeutic applications for various disorders and trauma. It is important, however, to keep in mind the difference between the embryonic stem cells, somatic stem cells and somatic precursor cells when considering potential clinical applications. Here we provide the review of the current status of stem cell field and discuss the potential of therapeutic applications for blood and Immune system disorders, multiple sclerosis, hypoxic-ischemic brain injury and brain tumors. For the complimentary information about various stem cells and their properties we recommend consulting the National Institutes of Health stem cell resources (http://stemcells.nih.gov/info/basics).
Assuntos
Células-Tronco , Adulto , Animais , Embrião de Mamíferos/citologia , Rejeição de Enxerto , Humanos , Neoplasias/patologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
We report on the size distribution of clones marked by mitotic recombination induced by several different doses of X-rays applied to 72 h oldDrosophila larvae. The results indicate that the radiation significantly reduces the number of cells which undergo normal proliferation in the imaginal wing disc. We estimate that 1000 r reduces by 40-60% the number of cells capable of making a normal contribution to the development of the adult wing. Part of this reduction is due to severe curtailment in the proliferative ability of cells which nevertheless remain capable of adult differentiation; this effect is possibly due to radiation-induced aneuploidy. Cytological evidence suggests that immediate cell death also occurs as a result of radiation doses as low as 100 r. The surviving cells are stimulated to undergo additional proliferation in response to the X-ray damage so that the result is the differentiation of a normal wing.
RESUMO
Proteins of the 4.1 family play a key role in the integrity of the cytoskeleton and in epithelial tissue movement, as shown by the disruption of the actin cytoskeleton in human erythrocytes caused by genetic loss of protein 4.1, and the failure of epithelial tissue migration during Drosophila embryogenesis caused by genetic loss of the 4.1 homolog Coracle. Here we report the genetic characterization of Yurt, a novel protein 4.1 family member in Drosophila that is associated with the plasma membrane of epithelial cells. Homozygous loss-of-function mutations in the yurt gene cause failure of germ-band retraction, dorsal closure, and head involution, associated with degeneration of the amnioserosa and followed by embryonic lethality. A mammalian homolog of Yurt is up-regulated in metastatic melanoma cells. These novel cytoskeletal proteins appear to play important roles in epithelial cell movements and in the morphogenetic tissue changes that depend on them.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Epitélio/embriologia , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The mutationsvestigial (vg; recessive) andUltravestigial (vg U; dominant) ofDrosophila melanogaster give rise to identical mutant adult phenotypes in which much of the cases this results from cell death in the presumptive wing margin of the wing disc in the third larval instar, but the process of cell degeneration is quite different in the two mutants. Invg cell death occurs continuously throughout the third larval instar, while invg U it occurs only in the early third instar. Cells fragment and some of the fragments condense, becoming electron dense ("apoptosis"). Both condensed and ultrastructurally normal cell fragments are extruded to the basal side of thevg disc epithelium. They accumulate under the basal lamina in the wing pouch area until they are phagocytosed by blood cells entering the wing pouch during the six hours following pupariation. Fragments are not extruded from thevg U epithelium but are apparently phagocytosed by neighboring epithelial cells. The basal lamina undergoes mophological changes following pupariation and is phagocytosed by blood cells in both wild-type andvestigial, but investigial the degenerated cell fragments are also engulfed by the same blood cells.
RESUMO
Human neural progenitor cells (hNPCs) can be recovered from postmortem human brains and used to study the molecular basis of neurogenesis. Human NPCs are being used to investigate the molecular basis of cell fate determination during stem cell divisions, based on comparison with the Drosophila model system. Drosophila neuroblasts and sensory organ precursors undergo well-defined asymmetric cell divisions (ACD), under the control of a genetically defined set of apical and basal determinants that are localized tightly and dynamically during division. We show by indirect immunofluorescence, confocal microscopy, and time-lapse video-microscopy that LGN and AGS3, two human homologs of the Drosophila ACD determinant Pins, have distinct patterns of localization in hNPCs. When cells are grown under conditions favoring proliferation, LGN is distributed asymmetrically in a cell cycle-dependent manner; it localizes to one side of the dividing cell and segregates into one of the daughter cells. When the cells are grown under conditions favoring differentiation, LGN accumulates in double foci similar to those containing the mitotic apparatus protein NuMA, and in a pattern shown previously for LGN and NuMA in differentiated cells. AGS3, a slightly more distant Pins homolog than LGN, does not show asymmetric localization in these cells. The progenitor cell marker nestin also localizes asymmetrically in colcemid-treated hNPCs and colocalizes with LGN. The results suggest that hNPCs undergo ACD and that similar molecular pathways may underlie these divisions in Drosophila and human cells.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Drosophila/química , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Células-Tronco/metabolismo , Homologia Estrutural de Proteína , Animais , Antígenos Nucleares , Encéfalo/citologia , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Demecolcina/farmacologia , Drosophila , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica/métodos , Proteínas de Filamentos Intermediários/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Nestina , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/metabolismo , Fatores de Tempo , Transfecção/métodos , Tubulina (Proteína)/metabolismo , Gravação de Videodisco/métodosRESUMO
We describe a protein family in Drosophila containing six adenosine deaminase-related growth factors (ADGFs), which are homologous to a mitogenic growth factor discovered in conditioned medium from cells of a different fly species, Sarcophaga. Closely related proteins have been identified in other animals, and a human homolog is implicated in the genetic disease Cat-Eye Syndrome. The two most abundantly expressed ADGFs in Drosophila larvae are ADGF-A, which is strongly expressed in the gut and lymph glands, and ADGF-D, which is mainly expressed in the fat body and brain. Recombinant ADGF-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum-independent proliferation of imaginal disk and embryonic cells in vitro. The enzymatic activity of these proteins is required for their mitogenic function, making them unique among growth factors. A culture medium prepared without adenosine, or depleted of adenosine by using bovine ADA, also stimulates proliferation of imaginal disk cells, and addition of adenosine to this medium inhibits proliferation. Thus ADGFs secreted in vivo may control tissue growth by modulating the level of extracellular adenosine.
Assuntos
Adenosina/metabolismo , Proteínas de Drosophila/fisiologia , Adenosina Desaminase/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Divisão Celular , Drosophila , Proteínas de Drosophila/genética , Dados de Sequência MolecularRESUMO
Post-mortem human brain tissue represents a vast potential source of neural progenitor cells for use in basic research as well as therapeutic applications. Here we describe five human neural progenitor cell cultures derived from cortical tissue harvested from premature infants. Time-lapse videomicrography of the passaged cultures revealed them to be highly dynamic, with high motility and extensive, evanescent intercellular contacts. Karyotyping revealed normal chromosomal complements. Prior to differentiation, most of the cells were nestin, Sox2, vimentin, and/or GFAP positive, and a subpopulation was doublecortin positive. Multilineage potential of these cells was demonstrated after differentiation, with some subpopulations of cells expressing the neuronal markers beta-tubulin, MAP2ab, NeuN, FMRP, and Tau and others expressing the oligodendroglial marker O1. Still other cells expressed the classic glial marker glial fibrillary acidic protein (GFAP). RT-PCR confirmed nestin, SOX2, GFAP, and doublecortin expression and also showed epidermal growth factor receptor and nucleostemin expression during the expansion phase. Flow cytometry showed high levels of the neural stem cell markers CD133, CD44, CD81, CD184, CD90, and CD29. CD133 markedly decreased in high-passage, lineage-restricted cultures. Electrophysiological analysis after differentiation demonstrated that the majority of cells with neuronal morphology expressed voltage-gated sodium and potassium currents. These data suggest that post-mortem human brain tissue is an important source of neural progenitor cells that will be useful for analysis of neural differentiation and for transplantation studies.