RESUMO
The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.
Assuntos
Terapia Genética , Proteínas de Filamentos Intermediários/fisiologia , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiopatologia , Retinose Pigmentar/fisiopatologia , Retinose Pigmentar/terapia , Animais , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Terapia Genética/métodos , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Periferinas , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Rodopsina/metabolismoRESUMO
Serum autoantibodies were induced in Lewis rats by immunization with a mixture of lentil lectin-binding glycoproteins isolated from bovine brain myelin. Intraperitoneal administration of 2-10 million syngeneic myelin basic protein-activated spleen cells to these rats led within 4-5 days to paralysis which, in most cases, persisted for several weeks. The major neuropathological features of the disease were numerous macrophages in both brain and spinal cord and large areas of demyelination, generally with axon preservation, particularly adjacent to the pial surfaces of the cord. This model is easily induced and will be useful for studies of demyelination and remyelination.
Assuntos
Antígenos/imunologia , Autoanticorpos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Glicoproteínas/imunologia , Lectinas de Plantas , Animais , Encéfalo/patologia , Química Encefálica , Proteínas de Transporte/imunologia , Bovinos , Encefalomielite Autoimune Experimental/patologia , Imunização , Lectinas/metabolismo , Macrófagos/patologia , Proteína Básica da Mielina/farmacologia , Bainha de Mielina/química , Ratos , Ratos Endogâmicos Lew , Medula Espinal/patologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/transplanteRESUMO
OBJECTIVE: To investigate the ultrastructural features of idiopathic full-thickness macular hole (FTMH) opercula excised during vitrectomy and to correlate them with the outcome of surgery. METHODS: Opercula were collected from eyes undergoing vitrectomy for stage 3 FTMH using noncrushing, cupped foreign body forceps. Following immediate fixation, specimens were processed for transmission electron microscopy. The ultrastructural features were correlated with the clinical data recorded for each patient before and after surgery. RESULTS: Eighteen specimens were studied. Native vitreous collagen was identified on the surface of all 18, while fragments of internal limiting membrane were present in 11 (61%). Eleven (61%) were found to contain only glia, comprising fibrous astrocytes and Müller cells in variable proportions. The remaining 7 (39%) were found to contain, in addition to glia, neurites and synaptic complexes, of which some were typical of cone photoreceptors. The initial surgical closure rate was significantly better in eyes in which only glia were present (9/11 [82%]), compared with those with neurites (1/7 [14%]) (P = .01). Once closure had been achieved with reoperation, the median final visual acuity was 20/60 in both groups (P = .26), although the likelihood of achieving an acuity of 20/40 or better was greater in the former (50%) than the latter group (17%). CONCLUSIONS: Two distinct types of opercula occur in association with stage 3 FTMH--those containing only glia (pseudo-opercula), which are probably associated with a foveal dehiscence and little or no loss of foveal tissue, and those containing both glia and a significant number of avulsed foveal cones (true opercula), which arise from a full-thickness foveal tear. Although the loss of foveal tissue in true opercula would seem to explain the worse initial anatomical and more modest visual results in some eyes, significant visual improvement may still be achieved after successful closure. The presence of neurites in true opercula suggests that, in at least some cases, direct traction on the foveal retina leads to macular hole formation.
Assuntos
Retina/ultraestrutura , Perfurações Retinianas/patologia , Idoso , Idoso de 80 Anos ou mais , Membrana Basal/ultraestrutura , Colágeno/ultraestrutura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neuroglia/ultraestrutura , Perfurações Retinianas/cirurgia , Acuidade Visual , Vitrectomia , Corpo Vítreo/ultraestruturaRESUMO
The effect of specific irreversible inhibitors of complexes I, III, IV and V of the mitochondrial respiratory chain, (rotenone, myxothiazol, cyanide and oligomycin, respectively) on mitochondrial N-acetylaspartate production, and its relationship to oxidative phosphorylation (ATP production and oxygen consumption) were investigated in isolated rat brain mitochondria. Mitochondrial N-acetylaspartate production, ATP production and oxygen consumption were all significantly decreased in the presence of each of the inhibitors used compared with control incubations, and correlated positively with each other. It is postulated that decreased N-acetylaspartate levels seen in disease states by 1H NMR spectroscopy in vivo may reflect primarily an impaired mitochondrial energy production rather than neuronal cell loss.
Assuntos
Ácido Aspártico/análogos & derivados , Encéfalo/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Mitocôndrias/efeitos dos fármacos , Análise de Variância , Animais , Ácido Aspártico/biossíntese , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Complexo I de Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mitocôndrias/metabolismo , NADH NADPH Oxirredutases/antagonistas & inibidores , ATPases Translocadoras de Prótons/antagonistas & inibidores , Prótons , Ratos , Ratos WistarRESUMO
PURPOSE: To study the smoothing effect of phototherapeutic keratectomy (PTK) using masking fluids as an adjunct to standard photorefractive keratectomy ablations. METHODS: Six fresh porcine corneas underwent -6.00, -10.00, and -15.00 D sphere ablations using the VISX Star excimer laser. Multizone treatments to a maximum 6.5 mm radially symmetrical bed were used with a fluence of 160 mJ/cm2. Three of the treatments were supplemented with a thin layer of balanced salt solution and 6 microm of full beam PTK. The corneas were examined by electron microscopy. RESULTS: Smoother treatment zones were apparent in corneas undergoing PTK following PRK. The effect was more marked at higher dioptric ablations. CONCLUSION: PTK may improve surface smoothness after PRK, especially for higher dioptric ablations.
Assuntos
Córnea/cirurgia , Córnea/ultraestrutura , Ceratectomia Fotorrefrativa/métodos , Animais , Lasers de Excimer , Microscopia Eletrônica de Varredura , Miopia/patologia , Miopia/cirurgia , Distribuição Aleatória , Refração Ocular , SuínosRESUMO
PURPOSE: This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse model of inherited retinal degeneration is p53 dependent. METHODS: A colony of p53-deficient rds mice were obtained by crossing homozygous rds mice with animals homozygous for a targeted disruption of the p53 gene and genotyping the offspring of the F1 cross. Both parental strains were on a BALB/c background. Age matched p53-deficient rds mice and controls (p53-deficient, rds and BALB/c mice), were sacrificed from day 1 to day 58 after birth. Eyes were paraffin-embedded and a modified terminal dUTP nick-end labeling (TUNEL) technique was used to detect the number of cells displaying DNA fragmentation within the sectioned retina. Eyes were also resin-embedded for semi-thin and ultra-thin sectioning. RESULTS: The peak in photoreceptor apoptosis, which occurs at 16 days in the rds mouse, was delayed by 3 days in p53-deficient rds mice. In addition, there was also a delay in the loss of photoreceptor cells between 16 and 26 days. However, absence of p53 did not prevent retinal degeneration in the rds mouse. The number of photoreceptor cells in p53-deficient rds mice at 35 days was very similar to that in the controls. CONCLUSIONS: We have demonstrated that absence of p53 delays but does not prevent photoreceptor cell loss in the rds mouse. Our results provide evidence for plasticity in the mechanism by which apoptosis proceeds in retinal degeneration.
Assuntos
Apoptose , Genes p53 , Células Fotorreceptoras de Vertebrados/patologia , Retina/patologia , Degeneração Retiniana/patologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Primers do DNA/química , Deleção de Genes , Genótipo , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retina/fisiopatologia , Retina/ultraestrutura , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologiaRESUMO
Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.
Assuntos
Resposta ao Choque Térmico/efeitos dos fármacos , Hidroxilaminas/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Retinose Pigmentar/prevenção & controle , Rodopsina/deficiência , Rodopsina/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eletrorretinografia , Humanos , Mutação , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Retinose Pigmentar/fisiopatologia , Rodopsina/genética , Fatores de Tempo , Transfecção , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Visão Ocular/efeitos dos fármacosRESUMO
The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre-existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex-mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex-mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/efeitos dos fármacos , Pseudópodes/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/isolamento & purificação , Animais , Plaquetas/química , Linhagem Celular Tumoral , Citoesqueleto/ultraestrutura , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Fluoresceína-5-Isotiocianato , Imunofluorescência , Corantes Fluorescentes , Glutationa Transferase/metabolismo , Humanos , Neoplasias Mamárias Experimentais/patologia , Microinjeções , Músculo Esquelético/química , Polímeros/química , Estrutura Terciária de Proteína , Coelhos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas , Proteína da Síndrome de Wiskott-Aldrich/químicaRESUMO
The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , HIV-1/genética , Epitélio Pigmentado Ocular/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Retinose Pigmentar/terapia , Animais , Eletrorretinografia , Humanos , Injeções , Microscopia Eletrônica , Modelos Animais , Células Fotorreceptoras/patologia , Epitélio Pigmentado Ocular/fisiopatologia , Epitélio Pigmentado Ocular/ultraestrutura , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Mutantes , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Vírus Formadores de Foco no Baço/genética , Fatores de Tempo , c-Mer Tirosina QuinaseRESUMO
A toxigenic strain of Escherichia coli displayed important structural modifications when placed in seawater which naturally lacked nutritive elements, as observed by electron microscopy. These include cell wall and cell body distortion, modification of the membranes, central segregation of the chromosome, and retraction of the cytoplasm. These modifications were accompanied by a decrease in cell protein content of approximately 40%. Certain cytoplasmic membrane proteins were lost, and new ones appeared. The development of these changes was considerably slower in cells that had previously been grown in a seawater medium. This suggests that osmotic regulation mechanisms, which enable E. coli to survive much longer in marine conditions, may have a protective influence.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/ultraestrutura , Água do Mar , Microbiologia da Água , Parede Celular/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Escherichia coli/crescimento & desenvolvimento , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Temperatura , Equilíbrio HidroeletrolíticoRESUMO
By use of experimental microcosms, it was demonstrated that the survival of Escherichia coli in nutrient-free seawater depended on the age of cells and on some physicochemical conditions during their prior growth. Cells grown in a bacteriological medium, with an acid or an alkaline pH, at high temperature (44 degrees C), or in the absence of oxygen were more sensitive to exposure to seawater of low nutrient content. In contrast, some complex media allowed production of cells adapting more rapidly to seawater. Cells grown in urine were far more sensitive than those grown in all bacteriological media tested. The sensitivity of all cells was highest when they were harvested during the early exponential phase of growth.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Água do Mar , Microbiologia da Água , Anaerobiose , Meios de Cultura , Humanos , Concentração de Íons de Hidrogênio , Cloreto de Sódio , Temperatura , Fatores de Tempo , UrinaRESUMO
A single dose (10 mg/kg) of Adriamycin was given to 23 adult Wistar rats and the spinal ganglia were studied from 6 h to 15 days after. As previously described, this drug results in the appearance of 'clear' areas in the nuclei of rat spinal ganglion cells as seen by light and by electron microscopy. The 'clear' areas become less conspicuous during the week before the onset of cytoplasmic degeneration. In addition, nucleolar changes become increasingly evident with time after injection. Fibrillar centres enlarge and nucleolar segregation is present from 24 h onwards, although the latter is invariably partial and more readily seen with the electron microscope. Nucleolar fragmentation is seen more frequently from 3 days onwards and nucleolar enlargement is common from 6 days. Early cytoplasmic abnormalities are associated with pronounced loss of Nissl substance. Adriamycin must bind extensively to nuclear DNA in spinal ganglion cells, causing the 'cleared' nuclear areas and the changes in dense chromatin. In addition, the binding of Adriamycin to nucleolar DNA with disturbance to nucleolar functions must be important in producing later cytoplasmic changes that precede cell death. There are thus similarities between the action of Adriamycin on these cells and those of Cisplatin, although in the latter intoxication the nuclear effects are significantly less prominent.
Assuntos
Doxorrubicina/toxicidade , Gânglios Espinais/efeitos dos fármacos , Animais , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/ultraestrutura , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Feminino , Gânglios Espinais/ultraestrutura , Microscopia Eletrônica , Ratos , Ratos Endogâmicos , Coloração e RotulagemRESUMO
Some metabolic modifications of Escherichia coli cells during starvation in seawater were studied in laboratory microcosms. The apparent die-off of this bacterium under such conditions, as observed by comparing the enumeration of CFU in conventional freshwater media and direct epifluorescence counts, was partially prevented when cells were previously grown in salted organic medium or on seawater-wastewater agar. beta-Galactosidase activity of starved cells disappeared gradually with time, even though some other enzymatic activities, such as that of alkaline phosphatase, increased. Moreover, some modifications of sensitivity to antibiotics, heavy metals, and bacteriophages in seawater- and wastewater-grown cells suggested that the cells undergo structural changes under natural marine conditions. These results provide additional experimental data indicating the possible active adaptation of E. coli cells to seawater.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Água do Mar , Microbiologia da Água , Fosfatase Alcalina/metabolismo , Antibacterianos/farmacologia , Meios de Cultura , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Esgotos , beta-Galactosidase/metabolismoRESUMO
This study compared the effects of sea water on Vibrio cholerae and six other Vibrio spp. Survival in seawater microcosms as well as uptake of a carbonated substrate in marine or non-marine conditions were investigated. Except for V. vulnificus becoming non-culturable, all the other selected species survived in sea water for at least 15 days at 20 degrees C. Depending on the species tested, the substrate was better transported in a high salt medium (V. cholerae, V. fluvialis and V. metschnikovii), than in a low salt medium (V. fluvialis, V. furnissii, V. parahaemolyticus and V. vulnificus). In terms of the response of the species to marine conditions, no correlation was found between survival in sea water and substrate uptake.
Assuntos
Água do Mar , Vibrio cholerae/crescimento & desenvolvimento , Vibrio/crescimento & desenvolvimento , Microbiologia da Água , Meios de Cultura , Metilglucosídeos/metabolismo , Cloreto de Sódio/metabolismo , Vibrio/metabolismo , Vibrio cholerae/metabolismoRESUMO
Important questions remain unanswered about the sequence of events leading to progressive and ultimately irreversible tissue damage in MS. This study was designed to investigate the pathological characteristics of, and function of, the blood-brain barrier within longstanding MS lesions using quantitative and Gd-DTPA enhanced MRI techniques. The ultrastructural appearances of postmortem lesions from a single, separate case of MS have been correlated with the MRI findings. Both MRI and ultrastructural analysis revealed considerable heterogeneity in the chronic lesions: some are 'closed' with no detectable extracellular water, but most are 'open' and show expansion of the extracellular space to as much as 87% of tissue area. This variable expansion probably results from differing degrees of axonal loss. Evidence of blood-brain barrier damage was found in only 17% of lesions, was less severe than that seen in acute lesions, and may result from repeated previous inflammatory insults. The findings imply progressive axonal loss in lesions as they age. It is possible that this loss is related to clinical progression of the disease.
Assuntos
Encéfalo/patologia , Esclerose Múltipla/patologia , Adulto , Encéfalo/ultraestrutura , Feminino , Gadolínio , Gadolínio DTPA , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Microscopia Eletrônica/métodos , Compostos Organometálicos , Ácido PentéticoRESUMO
In filtered natural seawater supplemented with potassium glutamate, the ability of Escherichia coli MC4100 cells to grow on a complex medium was enhanced as a logarithmic function of the external glutamate concentration. By comparison, a glutamate-respiring strain of E. coli exhibited a greater decline in culturability in seawater, suggesting a protective influence of the accumulated amino acid. Potassium glutamate increased the uptake of 14C-glycinebetaine by E. coli MC4100 cells in seawater and enhanced the protective effects of the betaine against culturability loss, possibly by increasing the expression of the ProU transport system. This bacterium apparently was able to synthesize glutamate because a protective effect (i.e. a lower culturability loss) was observed in seawater when supplemented with precursor compounds (2-oxoglutarate and glutamine). The combination of 2-oxoglutarate and glutamine resulted in the greatest protection of cells, possibly due to the synthesis of glutamate through glutamine 2-oxoglutarate amino transferase activity. The possible influence of glutamate and its precursors on survival of E. coli cells in the natural marine environment is considered, since glutamate, glutamine and betaines have been found in marine coastal waters and sediments.
Assuntos
Betaína/metabolismo , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Glutamatos/metabolismo , Microbiologia da Água , Transporte Biológico , Contagem de Colônia Microbiana , Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Água do MarRESUMO
Two types of glial cells occur in the retina, Müller cells and astrocytes. These cells share several structural features such as extending endfeet onto blood vessels of the retina. Retinal vessels express a tight blood-retinal barrier which is comparable to the blood-brain barrier (BBB) of the CNS. While astrocytes have been implicated in the induction of the BBB, the role of Müller cells in the blood-retinal barrier is unknown. To determine if Müller cells are capable of influencing vascular permeability, we have prepared Müller cells that are free of astrocytes and transplanted them to a peripheral target, the anterior eye chamber. Müller cells were identified 2 weeks to 3 months after injection and were predominantly localized within the connective tissue of the ciliary body. The Müller cells occurred as dense clusters of cells closely associated with ciliary blood vessels. The ciliary vessels adjacent to Müller cells were freely permeable to circulating horseradish peroxidase (HRP), suggesting that Müller cells did not induce tight barrier properties from these leaky peripheral vessels. In contrast, cortical astrocytes injected into the anterior eye chamber preferentially formed a monolayer on the anterior surface of the iris, a region known to contain blood vessels that are impermeable to circulating tracers (e.g., Raviola, Exp Eye Res [Suppl] 25:27, 1977). Müller cells were rarely associated with the iris and the few cells that were present were located deep within the iris stroma rather than on the surface. The behaviour of guinea pig Müller cells transplanted to the anterior eye chamber contrasts sharply with that of cortical astrocytes in terms of: 1) the ocular compartment to which Müller cells migrate; 2) the tissue invasiveness of the cells; and 3) the degree of permeability of blood vessels adjacent to transplanted cells. The results of this study emphasize the functional distinctness of the two types of retinal glia and suggest that Müller cells from guinea pig retina may not be active in modifying the permeability properties of peripheral blood vessels, a function that has been suggested for astrocytes.
Assuntos
Retina/fisiologia , Retina/transplante , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais , Astrócitos/citologia , Astrócitos/fisiologia , Permeabilidade Capilar , Células Cultivadas , Proteína Glial Fibrilar Ácida/análise , Cobaias , Imuno-Histoquímica , Queratinas/análise , Retina/citologia , Vasos Retinianos/fisiologia , Vimentina/análiseRESUMO
Using strains with or without the PhoE porin or different components of the phosphate regulon, we determined that maintenance of the culturability of Escherichia coli in seawater depended significantly on the presence of structures allowing access of phosphate ions to the periplasm, then to the cytoplasm of cells. Cells totally deprived of the two main phosphate transport systems (Pit, Pst) exhibited the highest loss of culturability. Most of this effect resulted from the loss of the high-affinity Pst system, and more specifically that of the periplasmic phosphate-binding protein PhoS. Survival was enhanced in seawater supplemented with phosphate (0.5 mM), whether or not these structures were present. From an ecological point of view, it is assumed that the presence of phosphate ions, even at low concentrations, can influence the behavior of E. coli cells in seawater.