RESUMO
In human erythrocyte membranes, various influx and efflux transporters are functionally expressed. However, their transport characteristics and modulation under disease states are not fully understood. In this study, we first examined the expression and detailed transport characteristics of breast cancer resistance protein (BCRP), an efflux ABC transporter, using inside-out membrane vesicles (IOVs) prepared from human erythrocytes, and then studied the effect of membrane cholesterol on BCRP function. The expression of BCRP was confirmed by western blotting; most of them being homodimers. The uptake of lucifer yellow (LY), a fluorescent BCRP substrate, into IOVs was time-, temperature-, and ATP-dependent, and the concentration of ATP which induced half-maximal stimulation of LY uptake was calculated to be 0.39 mM. The uptake of LY by IOVs was saturable with a Km value of 166 µM, and was inhibited by various BCRP inhibitors and substrates, such as fumitremorgin C and mitoxantrone. When membrane cholesterol content was increased by treating IOVs with cholesteryl hemisuccinate, LY uptake decreased with increasing cholesterol content. These results suggest that transport activity of BCRP in human erythrocyte membranes may be suppressed under disease states, such as hypercholesterolemia, that increase membrane cholesterol content.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Colesterol/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de Neoplasias/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico/fisiologia , Western Blotting , Regulação da Expressão Gênica , Humanos , Indóis/farmacologia , Isoquinolinas/metabolismo , Mitoxantrona/farmacologia , Proteínas de Neoplasias/metabolismo , Temperatura , Fatores de TempoRESUMO
The Pentax Airway Scope(®) is a single-use optical videolaryngoscope designed to assist with difficult tracheal intubation. We systematically reviewed the efficacy of the Pentax Airway Scope with that of a conventional laryngoscope for tracheal intubation in adults with 'normal' and 'difficult' airways. We included 17 randomised controlled trials with a total of 1801 participants. We used the DerSimonian and Laird random-effects model to calculate pooled relative risk or weighted mean differences. The relative risk (95% CI) of a Cormack-Lehane grade-1 laryngeal view was 2.40 (1.76-2.49) with the Pentax Airway Scope compared with the Macintosh laryngoscope, p < 0.00001. We found no other differences between the two laryngoscopes. Despite a superior laryngeal view, the Pentax Airway Scope provides little clinical benefit over the conventional laryngoscope.
Assuntos
Intubação Intratraqueal/instrumentação , Laringoscópios , Adulto , HumanosRESUMO
Many techniques have been proposed for esophageal reconstruction after esophagectomy when a gastric tube cannot be employed. There are two essential criteria for such a substitute: substitute length and sufficient blood supply. We propose ileocolic interposition as an easy and safe option. Two technical aspects contributing to the high success rate of this method are the preservation of an intact arterial network allowing normal blood flow to the ileocolic area, and the ability to quantify blood flow using a Doppler pulse flow meter in six cases. These are enabled by a long (up to 20cm) ileocolic segment. The preservation of the right colic artery is important, because its interruption would reduce blood supply to the long ileum segment. Between July 2003 and October 2008, we used this method in six patients in whom a gastric tube was not an option. We assessed perioperative morbidity and swallowing difficulties in each patient, quantifying dysphagia on scale of 0 to 4. There was no mortality and no anastomotic leak. There was one wound infection, and in one patient, recurrent nerve paralysis was observed. The postoperative hospital stay was 29.5 ± 10.8 days. The average dysphagia score for the six patients was 0.17 ± 0.41 after the operation. All patients can eat normally, without any dietary limitations. Ileocolonic interposition after esophagectomy requires careful assessment of the vascular supply. In this small series, morbidity was low and there was no perioperative mortality. We believe that this is an easy and safe method of reconstruction after esophagectomy in cases in whom a gastric tube cannot be used as a substitute.
Assuntos
Colo Ascendente/transplante , Esofagoplastia/métodos , Íleo/transplante , Fluxo Sanguíneo Regional , Idoso , Velocidade do Fluxo Sanguíneo , Carcinoma de Células Escamosas/cirurgia , Colo Ascendente/irrigação sanguínea , Transtornos de Deglutição/etiologia , Neoplasias Esofágicas/cirurgia , Esofagoplastia/efeitos adversos , Esôfago/lesões , Esôfago/cirurgia , Humanos , Íleo/irrigação sanguínea , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Ruptura/cirurgia , Índice de Gravidade de Doença , Ultrassonografia Doppler de PulsoRESUMO
The mechanism involved in generating anti-DNA antibodies (Abs) remains unclear, as DNA is poorly immunogenic. Molecular mimicry between DNA and non-DNA substances has been implicated as a possible mechanism. We previously reported that homocysteine-inducible endoplasmic reticulum protein (Herp), which is induced by endoplasmic reticulum stress, is recognized by anti-double-stranded DNA (dsDNA) IgG from patients with systemic lupus erythematosus and that immunization with Herp elicits anti-dsDNA Abs in BALB/c mice. In this study, we observed that anti-single-stranded DNA (ssDNA) Abs were also generated in Herp-immunized BALB/c mice and established an anti-Herp monoclonal antibody (mAb), HT4, which specifically cross-reacted with ssDNA. The epitope of the HT4 mAb on Herp, 'EPAGSNR', was identified by screening a synthetic peptide library. The binding of the HT4 mAb to the peptide was competitively inhibited by ssDNA. Immunization of the epitope peptide elicited anti-ssDNA Abs in BALB/c mice. These results indicate that the epitope exists in a human self-protein, mimics ssDNA and shows antigenicity for anti-ssDNA Abs in normal mice. Anti-ssDNA Abs are often found in patients with drug-induced lupus erythematosus. Treatment with representative drugs that cause drug-induced lupus (chlorpromazine, procainamide and hydralazine) induced Herp expression and apoptosis in HeLa cells. These findings suggest that molecular mimicry between Herp and ssDNA is involved in anti-ssDNA Ab production in drug-induced lupus.
Assuntos
Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , DNA de Cadeia Simples/imunologia , DNA/imunologia , Lúpus Eritematoso Sistêmico/induzido quimicamente , Proteínas de Membrana/imunologia , Animais , Anticorpos Antinucleares/biossíntese , Anticorpos Monoclonais/imunologia , Apoptose , Linhagem Celular Tumoral , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/imunologia , Células HeLa , Homocisteína/imunologia , Humanos , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de FluorescênciaRESUMO
OBJECTIVES: The aim of this study was to determine whether significant cranial and maxillary deformity exists in BALB/c-bm/bm (brachymorphism) mouse with spontaneous malocclusion using three-dimensional (3D) images. MATERIALS AND METHODS: Thirty female mice were divided into the following three groups: control group (BALB/c mice, n = 10), Norm group (BALB/c-bm/bm mice with normal occlusion, n = 10), and Mal group (BALB/c-bm/bm mice with malocclusion, n = 10). Nine points in the skull were selected, and transverse and antero-posterior distances were measured using three-dimensional images of micro-computed tomography (CT). Moreover, 3D images were superimposed at the median plane to visualize the skull shape asymmetry. RESULTS: The transverse distances at the posterior cranial and maxillary region and the antero-posterior distances in the Norm and Mal groups were significantly shorter than those in the control group. The nasal septum of the Mal group was significantly shorter than that of the Norm group. Morphological measurements and superimposed 3D images showed that lateral deviation occurred at the anterior cranial and maxillary region in the Mal group. CONCLUSION: The 3D micro-CT images revealed that the antero-posterior length and posterior transverse width at the cranium and maxilla in BALB/c-bm/bm mice were significantly smaller than those in BALB/c mice. It was quantitatively and morphologically clear that BALB/c-bm/bm mice show a spontaneous transverse crossbite owing to lateral deviation of the maxilla and nasal bone.
Assuntos
Cefalometria/métodos , Craniossinostoses/patologia , Imageamento Tridimensional/métodos , Má Oclusão/patologia , Maxila/patologia , Crânio/patologia , Microtomografia por Raio-X/métodos , Processo Alveolar/patologia , Animais , Suturas Cranianas/patologia , Feminino , Forame Magno/patologia , Osso Frontal/patologia , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Osso Nasal/patologia , Septo Nasal/patologia , Osso Occipital/patologia , Osso Parietal/patologia , Osso Temporal/diagnóstico por imagem , Zigoma/patologiaRESUMO
In mammalian cells, there are two major classes of sphingolipids---sphingomyelin and glycosphingolipids (GSLs)--both of which are synthesized from the hydrophobic molecule ceramide. The synthesis of most GSLs begins with glucosylation of ceramide to form glucosylceramide (GlcCer), which, in turn, serves as the source of 300-400 GSLs. Although most of these GSLs have been characterized chemically, the biological functions of ceramide glycosylation and GSLs still remain enigmatic. The recent description of a GSL-deficient cell line and isolation of cDNA for GlcCer synthase provide new insights into GSL functions.
Assuntos
Glucosiltransferases/metabolismo , Glicoesfingolipídeos/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Glucosiltransferases/química , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Glicosilação , Humanos , Dados de Sequência MolecularRESUMO
Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.
Assuntos
Glicoesfingolipídeos/metabolismo , Complexo de Golgi/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana , Proteínas de Neoplasias/metabolismo , Oxirredutases , Esfingosina/análogos & derivados , Animais , Sítios de Ligação , Células CHO , Bovinos , Membrana Celular/metabolismo , Cricetinae , Ativação Enzimática , Glucosiltransferases/genética , Levodopa/biossíntese , Lisossomos/metabolismo , Melaninas/metabolismo , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Pigmentação , Transporte Proteico , Proteínas/metabolismo , Psicosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Células Tumorais Cultivadas , Vacúolos/metabolismoRESUMO
OBJECTIVE: To evaluate the performance of the Airtraq optical laryngoscope for tracheal intubation by novice laryngoscopists, compared with that of the Macintosh laryngoscope. METHODS: Under supervision by staff anaesthetists, non-anaesthesia physicians performed tracheal intubation using either the Airtraq optical laryngoscope (n = 100) or the Macintosh laryngoscope (n = 100). The time required for airway instrumentation, the number of attempts until successful intubation and erroneous oesophageal intubation were investigated. RESULTS: The time to secure the airway was shorter with the Airtraq optical laryngoscope than with the Macintosh laryngoscope (p<0.001). The number of attempts until successful intubation was smaller with the Airtraq optical laryngoscope than with the Macintosh laryngoscope (p<0.001). Erroneous oesophageal intubation was less with the Airtraq optical laryngoscope than with the Macintosh laryngoscope (p<0.01). CONCLUSION: The Airtraq optical laryngoscope reduces the time to secure the airway and the incidence of failed tracheal intubation by novice laryngoscopists.
Assuntos
Educação de Pós-Graduação em Medicina , Intubação Intratraqueal/instrumentação , Laringoscópios , Competência Clínica/normas , Desenho de Equipamento , Humanos , Intubação Intratraqueal/métodos , Intubação Intratraqueal/normas , Pessoa de Meia-IdadeRESUMO
The aim of this study was to analyse hypoxia-associated dendritic cells (DCs) in colitic mice and the effects of probiotics on interleukin (IL)-10 production in inflammatory DCs under hypoxic conditions. Extensive hypoxia was observed in the colonic mucosa of dextran sodium sulphate-induced colitic mice. Flow cytometric analysis demonstrated that hypoxia-inducible factor-1α+ DCs in colonic lamina propria (CLP) lymphocytes and mesenteric lymph nodes (MLN) were more abundant in colitic mice than those in controls. Among three subsets of DCs, i.e. plasmacytoid DCs, conventional DCs (cDCs), and monocyte-derived DCs (mDCs), cDCs and mDCs were more abundant in CLP of colitic mice. Bone marrow-derived Flt-3L-induced DCs (Flt-DCs) but not bone marrow-derived GM-CSF-induced DCs (GM-DCs), incubated with 1% O2 exhibited an inflammatory phenotype, with higher CD86, IL-6, and tumour necrosis factor-α expression, and lower IL-10 levels than those in Flt-DCs incubated with 21% O2. The hypoxia-induced decrease in IL-10 expression in Flt-DCs was restored by Bifidobacterium bifidum JCM 1255T promoted IL-10 expression through the p38 pathway under normoxic conditions. The anti-inflammatory effects of B. bifidum JCM 1255T in Flt-DCs were mediated through different cellular mechanisms under hypoxic and normoxic conditions. B. bifidum JCM 1255T could be used therapeutically for its anti-inflammatory effects.
Assuntos
Células Dendríticas/patologia , Hipóxia/imunologia , Inflamação , Interleucina-10/biossíntese , Oxigênio/metabolismo , Probióticos , Animais , Diferenciação Celular , Células Cultivadas , Colite/induzido quimicamente , Colo/patologia , Células Dendríticas/imunologia , Feminino , Hipóxia/induzido quimicamente , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Mucosa/patologiaRESUMO
The Airtraq laryngoscope has an oropharyngeal airway-shaped blade that provides a non-line-of-sight view of the glottis. The configuration of the blade should mean that less movement of the cervical spine is required during laryngeal visualisation. We compared the degree of cervical spine movement in laryngoscopy performed using the Airtraq and conventional Macintosh laryngoscope. In 20 patients requiring general anaesthesia and tracheal intubation, we measured cervical spine movement using radiography in the same patient during consecutive procedures using the two laryngoscopes. Although significant movement of the cervical spine from baseline was noted during all procedures (p < 0.05), cervical spinal extension with the Airtraq was 29% less than that measured during Macintosh laryngoscopy between the occiput and C4, and 44% less at the C3/C4 motion segment (p < 0.05). Anterior deviations of the vertebral bodies from baseline were 32%, 35%, 38% and 40% less at the atlas, C2, C3, and C4 vertebrae, respectively, during Airtraq laryngoscopy than those measured during Macintosh laryngoscopy (p < 0.01). Our study demonstrated that laryngoscopy using the Airtraq laryngoscope involves less movement of the cervical spine compared to conventional procedures using a Macintosh laryngoscope.
Assuntos
Vértebras Cervicais/fisiologia , Laringoscópios , Movimento , Adulto , Anestesia Geral , Vértebras Cervicais/diagnóstico por imagem , Feminino , Movimentos da Cabeça , Humanos , Intubação Intratraqueal/instrumentação , Intubação Intratraqueal/métodos , Laringoscopia , Pessoa de Meia-Idade , RadiografiaRESUMO
Anti-DNA antibodies play an essential role in the pathogenesis of lupus nephritis. Mammalian DNA alone, however, is poorly immunogenic. We speculated that the antigenic trigger for the production of human nephritogenic anti-DNA antibodies is a non-DNA substance. The cDNA library from peripheral blood lymphocytes (PBLs) of a patient with active lupus nephritis was screened using the single-chain Fv of a human monoclonal nephritogenic O-81 anti-DNA antibody in a two-hybrid system. A clone containing the gene of an endoplasmic reticulum (ER) stress-inducible protein, Herp, was obtained: The O-81 antibody bound to recombinant Herp protein synthesized by Escherichia coli. Immunization with Herp elicited both anti-double-stranded DNA (anti-dsDNA) and anti-single-stranded (anti-ssDNA) antibodies in BALB/c mice and formed deposits of IgG in renal glomeruli. Anti-DNA antibodies purified from SLE sera bound to Herp. Moreover, anti-Herp antibodies showed specific binding to DNA. Herp was spontaneously expressed in PBLs of patients with active SLE, but not in PBLs of healthy subjects. These results imply that an inducible intracellular self-protein represents a candidate trigger for human nephritogenic anti-DNA autoantibodies. Any cell stress causing ER stress, such as viral infection, ultraviolet radiation, and chemicals, might be responsible for anti-DNA antibody production via Herp.
Assuntos
Anticorpos Antinucleares/imunologia , Autoantígenos/imunologia , Nefrite Lúpica/imunologia , Proteínas de Membrana/imunologia , Mimetismo Molecular/imunologia , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Feminino , Biblioteca Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro , Eritrócitos/citologia , Feminino , Fluoruracila/farmacologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Humanos , Interleucina-3/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Especificidade de Órgãos , RNA Mensageiro/análise , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/biossíntese , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Fator de Células-TroncoRESUMO
A mouse melanoma (B16) antigen was investigated at a cellular level by three blocking experiments using monoclonal antimelanoma antibodies, soluble melanoma antigen, and enzyme-treated B16 melanoma cells as inhibitors. The activity of antimelanoma cytotoxic T-lymphocytes (CTL) was specifically reduced by addition of the mixture of two monoclonal antimelanoma antibodies, one (M2590) recognizing the cross-species melanoma epitope on GM3(NeuAc) and the other (M562) reactive with the mouse melanoma-specific epitope on protein molecules. The CTL activity was also blocked by GM3 liposome as well as by the soluble antigen. However, 3,000 times more GM3 than the soluble melanoma antigen is required to obtain a similar inhibitory effect. When pronase-treated B16 melanoma cells, which have had protein molecules removed but GM3 left intact on the surface, were used as an inhibitor, their blocking activity was greatly reduced but was still partly observed at a high inhibitor/target ratio. These results indicate that the melanoma antigen is not GM3 itself but is composed of the GM3-protein complex. This finding was also supported by using an interleukin 2-dependent CTL clone whose activity was blocked by both M562 and M2590. Antimelanoma CTL were found to belong to a double-negative T-cell population with Thy-1+, Lyt-2-, L3T4- phenotypes. L3T4+ T-cells were also demonstrated to be necessary for induction of double negative antimelanoma CTL.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos Ly/imunologia , Antígenos de Neoplasias/análise , Melanoma Experimental/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Epitopos/análise , Gangliosídeo G(M3)/análise , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid (NeuGc)-containing gangliosides were isolated and characterized from human colon cancer tissues. The antigenic gangliosides were detected by thin-layer chromatography by our newly developed method (H. Higashi, Y. Fukui, S. Ueda, S. Kato, Y. Hirabayashi, M. Matsumoto, and M. Naiki. J. Biochem., 95: 1517-1520, 1984) of enzyme immunostaining using affinity-purified chicken antibody against hematoside containing NeuGc (II3NeuGc-LacCer) and horseradish peroxidase-conjugated rabbit anti-chicken lgG. One to six species of the antigenic gangliosides were isolated from seven of 16 cases of colon cancer, whereas no antigenic compound was detected in all apparently normal colorectal tissues from 17 individuals without colorectal cancer. Tissues from different patients showed different patterns of molecular species of the antigenic gangliosides. Densitometric determination indicated that HD antigenic sialic acid, NeuGc, accounted for about 1% or less of the total lipid-bound sialic acids. Four species of antigenic gangliosides were identified as hematoside and hematoside-containing O-acyl ester (II3NeuGc-LacCer and II3 4- or 7-O-acyl-NeuGc-LacCer), GM2-containing NeuGc (II3NeuGc-GgOse3Cer), and sialylparagloboside (IV3NeuGc-nLcOse4Cer) by their behaviors on 2-dimensional thin-layer chromatography, and the effects of mild alkaline treatment, sialidase treatment, periodate oxidation, and endo-beta-galactosidase treatment.
Assuntos
Antígenos Heterófilos/análise , Neoplasias do Colo/imunologia , Gangliosídeos/análise , Antígenos Heterófilos/isolamento & purificação , Densitometria , Gangliosídeo G(M2)/análise , Gangliosídeos/imunologia , Glicolipídeos/análise , HumanosRESUMO
l-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids d-serine and glycine. We have recently shown that l-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo l-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to l-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.
Assuntos
Encéfalo/enzimologia , Desidrogenases de Carboidrato/metabolismo , Neuroglia/enzimologia , Serina/biossíntese , Envelhecimento/metabolismo , Animais , Especificidade de Anticorpos , Astrócitos/citologia , Astrócitos/enzimologia , Encéfalo/citologia , Encéfalo/embriologia , Desidrogenases de Carboidrato/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Dendritos/enzimologia , Dendritos/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Neuroglia/citologia , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Bulbo Olfatório/enzimologia , Organelas/enzimologia , Organelas/ultraestrutura , Fosfoglicerato Desidrogenase , RNA Mensageiro/biossíntese , Células-Tronco/citologia , Células-Tronco/enzimologia , Sinapses/enzimologia , Sinapses/ultraestruturaRESUMO
An alpha-fucosyltransferase activity has been demonstrated in rat ascites hepatoma AH 7974F cells catalyzing the transfer of L-fucose to asialo-GM1 prepared from bovine brain GM1 ganglioside to form a fucolipid in the presence of Triton X-100. The radioactive fucolipid was shown to be Fuc-(alpha1 leads to 2)-Gal-(beta1 leads to 3)-GalNAc-(beta1 leads to 4)-Gal-(beta1 leads to 4)-Glc-ceramide from the following results. The radioactive product coincided with authentic blood group H-active fucolipid from AH 7974F cell on thin-layer chromatography. The product formed a precipitation line not only with Ulex europeus lectin but also with eel anti-H serum on agarose gel plates. The terminal 14C-labeled fucose was released by Bacillus fulminans alpha(1 leads to 2)fucosidase as well as Charonia lampas alpha-fucosidase. The optimum pH value for the incorporation of L-fucose into asialo-GM1 was 5.8 in cacodylate/HCl buffer. The fucosyltransferase was highly specific for asialo-GM1.
Assuntos
Fucose/metabolismo , Fucosiltransferases/metabolismo , Glicoesfingolipídeos/biossíntese , Hexosiltransferases/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Animais , Cálcio/farmacologia , Células Cultivadas , Detergentes/farmacologia , Gangliosídeo G(M1)/metabolismo , Glicoesfingolipídeos/metabolismo , Guanosina Difosfato Fucose/metabolismo , Concentração de Íons de Hidrogênio , Polietilenoglicóis/farmacologia , RatosRESUMO
The temporal change in the levels of the gangliotetraose-series gangliosides, i.e., GMla, GDla, GD1b, GT1b, in the blood plasma after intramuscular administration of bovine brain gangliosides (5 mg/kg) to beagle dogs (11.3-12.2 kg) was determined with high sensitivity by a recently developed thin-layer chromatography/enzyme-immunostaining method (Hirabayashi, Y., Koketsu, K., Higashi, H., Suzuki, Y., Matsumoto, M., Sugimoto, M. and Ogawa, T. (1986) Biochim. Biophys. Acta 876, 178-182). The amounts of GMla, GDla, GD1b, GT1b and their combined total in the plasma of beagle dogs before administration of gangliosides were 21 +/- 1, 36 +/- 7, 15 +/- 2, 16 +/- 2 and 88 +/- 6 pmol/ml of blood plasma, respectively. Trapezoidal calculation showed that the times of the maximum levels of GMla, GDla, GDlb, GTlb and the total of the their levels in the plasma were 8.0 +/- 1.2, 8.7 +/- 0.7, 6.3 +/- 2.0, 17.0 +/- 7.0 and 8.7 +/- 0.7 h after the administration of gangliosides, and their maximum concentrations were 517 +/- 37, 654 +/- 53, 160 +/- 5, 184 +/- 20 and 1383 +/- 74 pmol/ml, respectively. The maximum level of each ganglioside decreased gradually, reaching the normal level after 10 days. The half-maximum level of each ganglioside occurred 2-3 days after the administration. Asialo GM1 (GA1) was not detected plasma at any of the test times.
Assuntos
Química Encefálica , Gangliosídeos/sangue , Animais , Bovinos , Cromatografia em Camada Fina , Cães , Gangliosídeo G(M1)/sangue , Gangliosídeos/farmacologia , Técnicas ImunoenzimáticasRESUMO
alpha-N-Acetylgalactosaminidase, produced by Acremonium sp. destroyed blood group A active substance. This enzyme converted human erythrocytes from group A to O(H). The enzyme was ascertained to liberate N-acetylgalactosamine from group A erythrocytes membranes accompanying a decrease in hemagglutination inhibition activity of A erythrocyte-anti-A serum by the erythrocyte membranes. Glycoproteins and glycolipids extracted from A erythrocytes reduced their blood group A activity after the enzyme treatment. The enzyme also worked on Forssman hapten glycolipid.
Assuntos
Sistema ABO de Grupos Sanguíneos , Acremonium/enzimologia , Membrana Eritrocítica/efeitos dos fármacos , Hexosaminidases/farmacologia , Acetilgalactosamina/metabolismo , Membrana Eritrocítica/imunologia , Membrana Eritrocítica/metabolismo , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Testes de Inibição da Hemaglutinação , Hexosaminidases/isolamento & purificação , Humanos , alfa-N-AcetilgalactosaminidaseRESUMO
A N-glycolyneuraminic acid containing trisialoganglioside was isolated from bovine brains ganglioside mixture using Q-Sepharose. Its chemical structure was characterized as IV3NeuAc, II3NeuAc-NeuGc, Gg4Cer by gas-liquid chromatography, a permethylation study, sialidase degradation, TLC/enzyme-immunostaining, fast atom bombardment-mass spectrometry, fluorometric HPLC and proton nuclear magnetic resonance spectroscopy. This was unique in the mixed sialic acid constituents. (formula; see text) This accounted for 0.78% of the gangliosides. The ceramide structure was almost identical with those of major bovine brain ganglioside, as mainly composed of 18:0 fatty acid (90.9%) and d20:0 sphingosine base.
Assuntos
Química Encefálica , Bovinos/fisiologia , Gangliosídeos/química , Ácidos Neuramínicos/análise , Ácidos Siálicos/análise , Animais , Sequência de Carboidratos , Cromatografia em Camada Fina , Gangliosídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Metilação , Dados de Sequência Molecular , Neuraminidase/metabolismo , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
A comparative study of the glycolipids was performed on two cell lines of rat ascites hepatomas, island-forming and free cell types, and several marked differences were found as follows: 1. Hexose content in glycolipids derived from AH 7974F (free cell type) was about 3.6-fold as much as that from AH 7974 (island-forming cell type) on the basis of dry cell weight. 2. The glycolipids in the cells of AH 7974 were tentatively identified as glucosylceramide, lactosylceramide, galactosylgalactosylglucosylceramide, globoside and hematoside (GM3) by both thin-layer and gas-liquid chromatography. 3. On the other hand, the glycolipids in AH 7974F cells were glucosylceramide, lactosylceramide and at least four unknown lipids. 4. Structure of one of these unknown lipids was shown to be asialo-GM2 by methylation and enzymatic degradation studies. Moreover, the presence of asialo-GM1 was suggested by immunoprecipitation test with anti-asialo-GM1 serum. 5. Glucosylceramide and lactosylceramide from AH 7974F cells were found to possess hydroxy fatty acids as major fatty acids components, which were rare in these glycolipids from AH 7974 cells.