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Linear IgA bullous dermatosis (LABD) is an auto-immune disease affecting young children and adults, characterized by the linear deposition of IgA at the basement membrane zone with resultant complement activation and a cascade of immune reactions. There is a loss of adhesion at the dermo-epidermal junction and subsequent blister formation. It is a rare disease that has a good prognosis with adequate therapy. However, the underlying depressed immunity associated with the disease may expose them to such infections as tuberculosis. We report the case of an 11-years-old Nigerian female adolescent with LABD, diagnosed at the age of four years but defaulted on follow-up, who developed disseminated tuberculosis (pulmonary, lymph nodes, abdominal and pericardial effusion) seven years after the appearance of the initial blistering skin lesions. She commenced anti-tuberculosis drugs, steroids, and a tube pericardiostomy for the pericardial effusion. Dapsone was initiated for the LABD during the continuation phase of anti-tuberculosis therapy, with subsequent disappearance of the skin rash within two weeks.
La dermatose bulleuse linéaire à IgA (DBL) est une maladie auto-immune affectant les jeunes enfants et les adultes, caractérisée par le dépôt linéaire d'IgA dans la zone de la membrane basale, avec l'activation du complément qui en résulte et une cascade de réactions immunitaires. Il y a une perte d'adhérence à la jonction dermo-épidermique et une formation ultérieure de vésicules. C'est une maladie rare qui a un bon pronostic avec un traitement adéquat. Cependant, l'immunité déprimée sous-jacente associée à la maladie peut les exposer à des infections telles que la tuberculose. Nous rapportons le cas d'une adolescente nigériane de 11 ans atteinte de la LABD, diagnostiquée à l'âge de quatre ans mais en défaut de suivi, qui a développé une tuberculose disséminée (pulmonaire, ganglions lymphatiques, épanchement abdominal et péricardique) sept ans après l'apparition des lésions cutanées vésiculeuses initiales. Elle a commencé à recevoir des médicaments antituberculeux, des stéroïdes et une péricardiostomie par sonde pour l'épanchement péricardique. La dapsone a été initiée pour la DLB pendant la phase de continuation du traitement antituberculeux, avec une disparition de l'éruption cutanée en deux semaines. Mots clés: IgA linéaire, dermatose bulleuse, tuberculose disséminée, adolescent.
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Dermatose Linear Bolhosa por IgA , Derrame Pericárdico , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Imunoglobulina A/uso terapêutico , Dermatose Linear Bolhosa por IgA/diagnóstico , Dermatose Linear Bolhosa por IgA/tratamento farmacológico , Dermatose Linear Bolhosa por IgA/patologia , NigériaRESUMO
Ultrafast semiconductor disk lasers (SDLs) passively modelocked using semiconductor saturable absorbers mirrors (SESAMs) generate optical frequency combs (OFCs) with gigahertz line spacings - a regime where solid-state and fiber lasers struggle with geometrical and Q-switching limitations. We stabilized both the frequency comb spacing and the offset without any additional external optical amplification or pulse compression. The overall noise performance is competitive with other gigahertz OFCs. A SESAM-modelocked vertical external-cavity surface-emitting laser (VECSEL) at a center wavelength around 1 µm generates 122-fs pulses with 160 mW average output power and we only needed 17-pJ pulse energy coupled into a silicon nitride (Si3N4) waveguide for supercontinuum generation (SCG) and OFC offset stabilization.
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Little is known about emissions and exposure potential from vat polymerization additive manufacturing, a process that uses light-activated polymerization of a resin to build an object. Five vat polymerization printers (three stereolithography (SLA) and two digital light processing (DLP) were evaluated individually in a 12.85 m3 chamber. Aerosols (number, size) and total volatile organic compounds (TVOC) were measured using real-time monitors. Carbonyl vapors and particulate matter were collected for offline analysis using impingers and filters, respectively. During printing, particle emission yields (#/g printed) ranged from 1.3 ± 0.3 to 2.8 ± 2.6 x 108 (SLA printers) and from 3.3 ± 1.5 to 9.2 ± 3.0 x 108 (DLP printers). Yields for number of particles with sizes 5.6 to 560 nm (#/g printed) were 0.8 ± 0.1 to 2.1 ± 0.9 x 1010 and from 1.1 ± 0.3 to 4.0 ± 1.2 x 1010 for SLA and DLP printers, respectively. TVOC yield values (µg/g printed) ranged from 161 ± 47 to 322 ± 229 (SLA printers) and from 1281 ± 313 to 1931 ± 234 (DLP printers). Geometric mean mobility particle sizes were 41.1-45.1 nm for SLA printers and 15.3-28.8 nm for DLP printers. Mean particle and TVOC yields were statistically significantly higher and mean particle sizes were significantly smaller for DLP printers compared with SLA printers (p < 0.05). Energy dispersive X-ray analysis of individual particles qualitatively identified potential occupational carcinogens (chromium, nickel) as well as reactive metals implicated in generation of reactive oxygen species (iron, zinc). Lung deposition modeling indicates that about 15-37% of emitted particles would deposit in the pulmonary region (alveoli). Benzaldehyde (1.0-2.3 ppb) and acetone (0.7-18.0 ppb) were quantified in emissions from four of the printers and 4-oxopentanal (0.07 ppb) was detectable in the emissions from one printer. Vat polymerization printers emitted nanoscale particles that contained potential carcinogens, sensitizers, and reactive metals as well as carbonyl compound vapors. Differences in emissions between SLA and DLP printers indicate that the underlying technology is an important factor when considering exposure reduction strategies such as engineering controls.
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Poluição do Ar em Ambientes Fechados/análise , Material Particulado/análise , Impressão Tridimensional , Compostos Orgânicos Voláteis/análise , Carcinógenos , Metais , Tamanho da Partícula , Material Particulado/química , PolimerizaçãoRESUMO
We present the first direct carrier-envelope-offset (CEO) frequency detection of a modelocked laser based on supercontinuum generation (SCG) in a CMOS-compatible silicon nitride (Si(3)N(4)) waveguide. With a coherent supercontinuum spanning more than 1.5 octaves from visible to beyond telecommunication wavelengths, we achieve self-referencing of SESAM modelocked diode-pumped Yb:CALGO lasers using standard f-to-2f interferometry. We directly obtain without amplification strong CEO beat signals for both a 100-MHz and 1-GHz pulse repetition rate laser. High signal-to-noise ratios (SNR) of > 25 dB and even > 30 dB have been generated with only 30 pJ and 36 pJ of coupled pulse energy from the megahertz and gigahertz laser respectively. We compare these results to self-referencing using a commercial photonic crystal fiber and find that the required peak power for CEO beat detection with a comparable SNR is lowered by more than an order of magnitude when using a Si(3)N(4) waveguide.
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BACKGROUND: Chest pain may be the first symptom of developing respiratory malignancy, particularly in subjects with asbestos exposure, yet little information exists on this topic. AIMS: To investigate chest pain in a cohort of subjects exposed to asbestos and silica dust applying for compensation. METHODS: Cross-sectional study using a standardized questionnaire. Data collection included: smoking history, Medical Research Council scales of exercise capacity and respiratory symptoms. RESULTS: We studied 621 subjects. Six disease groups were categorized: asbestosis (n = 27), diffuse pleural thickening (DPT) (132), asbestosis and DPT (14), silicosis (26), pleural plaques only (160) and healthy subjects with a history of dust exposure (256). Crude prevalence rates of chest pain were high, with chest pain approximately twice as common in subjects with asbestos-related disorders and silicosis as in healthy subjects, with an overall frequency of ~40%. However, when other variables were taken into account in a multivariate analysis the differences between groups disappeared. The factor most significantly related to chest pain was age. CONCLUSIONS: Chest pain is apparently common in subjects with asbestos-related disorders and silicosis, but after adjustment for other variables, no increased prevalence was apparent in subjects with pleural disorders. More sophisticated questionnaires and dedicated imaging are required to elucidate this further.
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Amianto/toxicidade , Dor no Peito/epidemiologia , Pneumopatias/complicações , Doenças Profissionais/epidemiologia , Exposição Ocupacional/efeitos adversos , Dióxido de Silício/toxicidade , Idoso , Austrália/epidemiologia , Dor no Peito/etiologia , Poeira , Métodos Epidemiológicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Profissionais/etiologia , Doenças Pleurais/complicações , Inquéritos e QuestionáriosRESUMO
BACKGROUND: The Surveillance of Australian workplace Based Respiratory Events (SABRE) New South Wales (NSW) scheme is a voluntary notification scheme established to determine the incidence of occupational lung diseases in NSW Australia. AIMS: Data presented in this paper summarize the last 7 years of reporting to SABRE (June 2001 to December 2008). METHODS: Every 2 months, participating occupational physicians, respiratory physicians and general practitioners (accredited by the NSW WorkCover Authority) reported new cases of occupational lung disease seen in their practices. Data collected include gender, age, causal agent and the occupations and industries believed responsible. Estimated incidence was calculated for each disease. RESULTS: Three thousand six hundred and fifty-four cases were notified to the scheme, consisting of 3856 diagnoses. Most of the cases were males (76%). Pleural plaques [1218 (28%)] were the most frequently reported condition, followed by mesothelioma [919 (24%)]. Silicosis [90 (2%)] and occupational asthma [OA; 89 (2%)] were the most frequently reported non-asbestos-related diseases. Estimated rates for mesothelioma, diffuse pleural thickening (DPT) and OA were 83, 83 and 5 cases per million employed males per year, respectively. Trades such as carpenters and electricians associated with the building industry, electricity supply and asbestos product manufacture were the most common occupations and industries reported. CONCLUSIONS: Asbestos-related diseases are the most frequently reported conditions to SABRE NSW. The very low incidence of OA for NSW most likely reflects under-diagnosis as well as under-reporting. Occupational lung disease is still occurring in NSW despite current preventative strategies. The SABRE scheme currently provides the only available information in this area.
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Pneumopatias/epidemiologia , Doenças Profissionais/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , New South Wales/epidemiologia , Distribuição por Sexo , Adulto JovemRESUMO
Rats were raised on diets either rich or poor in strontium. Powder x-ray diffraction patterns suggest that isomorphous substitution of strontium for calcium occurs in the apatite of tooth enamel, and that strontium may form Sr(6)H(3)(PO(4)) * 2H(2)O, a compound hitherto unreported in biologic systems.
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Apatitas/metabolismo , Esmalte Dentário/metabolismo , Estrôncio/metabolismo , Animais , Cálcio/metabolismo , Masculino , Ratos , Difração de Raios XRESUMO
BACKGROUND: Emerging reports suggest the potential for adverse health effects from exposure to emissions from some additive manufacturing (AM) processes. There is a paucity of real-world data on emissions from AM machines in industrial workplaces and personal exposures among AM operators. METHODS: Airborne particle and organic chemical emissions and personal exposures were characterized using real-time and time-integrated sampling techniques in four manufacturing facilities using industrial-scale material extrusion and material jetting AM processes. RESULTS: Using a condensation nuclei counter, number-based particle emission rates (ERs) (number/min) from material extrusion AM machines ranged from 4.1 × 1010 (Ultem filament) to 2.2 × 1011 [acrylonitrile butadiene styrene and polycarbonate filaments). For these same machines, total volatile organic compound ERs (µg/min) ranged from 1.9 × 104 (acrylonitrile butadiene styrene and polycarbonate) to 9.4 × 104 (Ultem). For the material jetting machines, the number-based particle ER was higher when the lid was open (2.3 × 1010 number/min) than when the lid was closed (1.5-5.5 × 109 number/min); total volatile organic compound ERs were similar regardless of the lid position. Low levels of acetone, benzene, toluene, and m,p-xylene were common to both AM processes. Carbonyl compounds were detected; however, none were specifically attributed to the AM processes. Personal exposures to metals (aluminum and iron) and eight volatile organic compounds were all below National Institute for Occupational Safety and Health (NIOSH)-recommended exposure levels. CONCLUSION: Industrial-scale AM machines using thermoplastics and resins released particles and organic vapors into workplace air. More research is needed to understand factors influencing real-world industrial-scale AM process emissions and exposures.
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There is a paucity of data on additive manufacturing process emissions and personal exposures in real-world workplaces. Hence, we evaluated atmospheres in four workplaces utilizing desktop "3-dimensional" (3-d) printers [fused filament fabrication (FFF) and sheer] for production, prototyping, or research. Airborne particle diameter and number concentration and total volatile organic compound concentrations were measured using real-time instruments. Airborne particles and volatile organic compounds were collected using time-integrated sampling techniques for off-line analysis. Personal exposures for metals and volatile organic compounds were measured in the breathing zone of operators. All 3-d printers that were monitored released ultrafine and fine particles and organic vapors into workplace air. Particle number-based emission rates (#/min) ranged from 9.4 × 109 to 4.4 × 1011 (n = 9samples) for FFF3-d printers and from 1.9 to 3.8 × 109 (n = 2 samples) for a sheer 3-d printer. The large variability in emission rate values reflected variability from the printers as well as differences in printer design, operating conditions, and feedstock materials among printers. A custom-built ventilated enclosure evaluated at one facility was capable of reducing particle number and total organic chemical concentrations by 99.7% and 53.2%, respectively. Carbonyl compounds were detected in room air; however, none were specifically attributed to the 3-d printing process. Personal exposure to metals (aluminum, iron) and 12 different organic chemicals were all below applicable NIOSH Recommended Exposure Limit values, but results are not reflective of all possible exposure scenarios. More research is needed to understand 3-d printer emissions, exposures, and efficacy of engineering controls in occupational settings.
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We have tested whether the orientation of axons sprouting from bipolar dorsal root ganglion neurons is influenced by diffusible cues from surrounding tissues. Surface ectoderm, dermomyotome, and notochord exert strong chemorepulsion on axons growing in collagen gels, operating at separations beyond those found in vivo and active in cocultures of chick and mouse tissues. Basal and alar plates of the neural tube are devoid of activity, as is the posterior-half-sclerotome, which repels in a contact-dependent manner. When ganglia are sandwiched between dermomyotome and notochord placed at a distance, axon growth is channeled in a bipolar trajectory. These results show that gradients of diffusible repulsion molecules flanking axon pathways can generate linear patterns of axon growth. We suggest that such "surround repulsion" may function generally, in concert with contact-dependent guidance mechanisms, to guide axons in the developing nervous system.
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Gânglios Espinais/citologia , Neurônios Aferentes/citologia , Animais , Axônios/ultraestrutura , Embrião de Galinha , Colágeno , Técnicas de Cultura , Indução Embrionária , Músculos/embriologia , Notocorda/fisiologia , Pele/embriologiaRESUMO
Endothelial cells were cultured from various different human vessels, including aortas, pulmonary, ovarian, and umbilical arteries, and pulmonary, ovarian, and umbilical veins. The cultured cells were identified as endothelial cells by the presence of Factor VIII antigen and antiotensin I converting enzyme (kininase II). They retained these markers for at least five passages in culture, and some cells had them for seven passages or more. Endothelial cells from the various vessels were compared with respect to their ability to metabolize angiotensins I and II and bradykinin. Cells from arteries had three to five times the angiotensin I converting enzyme activity as cells from veins. The activity of angiotensinase A (aspartyl aminopeptidase) had a similar distribution, and cells from arteries were consistently more active than cells from veins. Cultures of endothelial cells from pulmonary and umbilical vessels formed prostacyclin in response to mechanical stimulation. Media from cell monolayers that were subjected to a change of medium and gentle agitation inhibited aggregation of human platelets. This inhibitory activity was generated within 2-5 min, and it was not formed by cells that were treated with indomethacin or tranylcypromine. Addition of prostaglandin (PG)H(2) to indomethacin-treated cells restored the ability to form the inhibitor, but cells treated with tranylcypromine were not responsive to PGH(2). In experiments where [(14)C]arachidonic acid was added to the cells before stimulation, the major metabolite identified by thin-layer chromatography was 6-keto PGF(1alpha). Thus, it appears that pulmonary endothelial cells, as well as umbilical cord cells, can form prostacyclin. In experiments comparing the ability of arterial and venous cells to form prostacyclin, arterial cells were more active than venous cells. These studies of cells from various human vessels suggest that the vascular origin of cultured endothelial cells determines how they metabolize vasoactive peptides and form prostacyclin.
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Pulmão/irrigação sanguínea , Adulto , Antígenos/análise , Aorta/citologia , Artérias/citologia , Células Cultivadas , Endotélio/citologia , Endotélio/metabolismo , Epoprostenol/biossíntese , Fator VIII/imunologia , Feminino , Humanos , Pessoa de Meia-Idade , Ovário/irrigação sanguínea , Agregação Plaquetária , Gravidez , Prostaglandinas/metabolismo , Artérias Umbilicais/citologia , Veias Umbilicais/citologia , Veias/citologiaRESUMO
Cultured endothelial cells provide a model for the study of interactions of vasoactive peptides with endothelium. Endothelial cell cultured from veins of human umbilical cords contain both angiotensin I converting enzyme (kininase II) and angiotensinase activities. Intact monolayers of cells can both activate angiotensin I and inactivate bradykinin when the peptides are added to culture flasks in protein-free medium. Intact suspended cells or lysed cells convert angiotensin I to angiotensin II, inactivate bradykinin, and hydrolyze hippuryldiglycine to hippuric acid and diglycine. These actions are inhibited by SQ 20881, the specific inhibitor of converting enzyme. The kininase activity of endothelial cells was partially inhibited by antibody to human lung converting enzyme. Endothelial cells also inactivate longer analogs of bradykinin, such as kallidin, methionyl-lysyl bradykinin, and bradykinin coupled covalently to 500,000 mol wt dextran. The endothelial cells retained converting enzyme activity through four successive subcultures, indicating that the enzyme is synthesized by the cells surface, and it is apparently a marker for endothelial cells, since cultured human fibroblasts, smooth muscle cells, and baby hamster kidney cells do not have it. Endothelial cells also contain an aminopheptidase which hydrolyzes both angiotensin II and the synthetic substrate, alpha-L-aspartyl beta-naphthylamide. The angiotensinase activity increased when the cells were lysed, which suggests that the enzyme is localized within the cells, Hydrolysis of both alpha-L-aspartyl beta-naphthylamide and angiotensin II was inhibited by omicron-phenanthroline, indicating that the enzyme is an A-tipe anigotensinase.
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Endopeptidases/metabolismo , Peptidil Dipeptidase A/metabolismo , Veias Umbilicais/enzimologia , Angiotensina II/metabolismo , Bradicinina/metabolismo , Cobalto/farmacologia , Ácido Edético/farmacologia , Endotélio/citologia , Endotélio/enzimologia , Feminino , Glicina/metabolismo , Hipuratos/metabolismo , Humanos , Pulmão/imunologia , Peptidil Dipeptidase A/imunologia , Fenantrolinas/farmacologia , Gravidez , Frações Subcelulares/enzimologia , Teprotida/farmacologiaRESUMO
Mitogen-stimulated human T cell activation is absolutely dependent on the participation of a nonresponding accessory cell. In populations of human peripheral blood mononuclear cells, monocytes function as the requisite accessory cells. The possibility that cultured endothelial cells (EC) might also function as accessory cells was studied by examining the potential of endothelial cells to restore mitogen responsiveness to monocyte-depleted human T cells. Highly purified T cells were prepared by isolating cells rosetting with sheep erythrocytes and removing monocyte contamination by glass adherence and nylon wool column passage. When cultured at low cell density, T cells failed to respond to stimulation with various mitogenic lectins, whereas co-culture with monocytes restored responsiveness. Similarly, EC obtained from umbilical vein, pulmonary artery, and ovarian vein restored the capacity of T cells to respond to mitogens. Mitogen-stimulated T cell activation required viable endothelial cells. Moreover, effective endothelial T cell cooperation appeared to involve the establishment of cell-to-cell contact between EC and responding T cells. Accessory cell function was not a nonspecific property of all tissue culture cells as evidenced by the finding that human foreskin fibroblasts, lung fibroblasts, and HeLa cells were unable to restore responsiveness to monocyte-depleted T cells. These observations indicate that endothelial cells can support the induction of mitogen-induced T cell activation and suggest that cells lining blood vessels may play an active role in the initiation of immune responses in vivo.
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Endotélio/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Células Cultivadas , Células HeLa/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/imunologia , Nitrosoguanidinas/farmacologia , Fito-Hemaglutininas/farmacologiaRESUMO
The activity of prolylcarboxypeptidase (PCP), or angiotensinase C, was measured in lung tissues, leukocytes, and cultured human cells using Cbz-Pro-[14C]Ala as a substrate. A lysosomal fraction of homogenized rat or human lung contained most of the PCP activity in that tissue. Polymorphonuclear neutrophils, macrophages, and lymphocytes isolated from human blood had PCP activity. Fibroblasts cultured from human tissues had the highest activity (0.56-1.15 mumol/h per 10(6) cells), more than endothelial cells cultured from human pulmonary arteries. PCP of cultured human fibroblasts was similar to the human renal enzyme because it was resistant to moderate heating and was not inhibited by p-chloromercuriphenyl sulfonic acid. These properties and the substrate specificity distinguish PCP from cathepsin A, which is also in fibroblasts. Antibody to human renal PCP reacted with fibroblast PCP in immunofluorescence, indicating common antigenic determinants. Hydrocortisone changed PCP activity in fibroblasts in parallel with changes in beta-glucuronidase activity and cell-protein concentration; the activity was depressed at low concentration of the hormone. PCP activity was also found in synovial fluid from arthritic joints and in fibroblasts from the synovium. That PCP is found in both inflammatory exudates and in cells that appear at sites of inflammation indicates that, in addition to inactivating angiotensins, this enzyme may have a role in inflammation.
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Carboxipeptidases/metabolismo , Pulmão/enzimologia , Angiotensina II/metabolismo , Animais , Células Cultivadas , Endotélio/enzimologia , Fibroblastos/enzimologia , Granulócitos/enzimologia , Substâncias de Crescimento/farmacologia , Humanos , Hidrocortisona/farmacologia , Linfócitos/enzimologia , Neutrófilos/enzimologia , Prolina/metabolismo , Ratos , Líquido Sinovial/enzimologiaRESUMO
Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [3H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (Mr greater than 5000) 3H-labeled glycopeptide. Digestion of these 3H-labeled glycopeptides with endo-beta-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid----Gal----GlcNAc----Gal. Treatment of [3H]glucosamine-labeled cells with melittin caused 3H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total 3H-labeled glycoproteins from the cell and 3% of the 3H-labeled proteoglycans. The 3H-labeled glycoproteins were digested with Pronase and the resulting 3H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (Mr greater than 5000) now comprised about 30% of these released 3H-labeled glycopeptides. These high molecular weight 3H-labeled glycopeptides were degraded with endo-beta-galactosidase but not with testicular hyaluronidase. Analysis of the released 3H-labeled glycoproteins indicated a preferential release of glycoproteins of 70-90 kDa enriched in lactosaminoglycan-type oligosaccharides.
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Amino Açúcares/biossíntese , Glicoproteínas/biossíntese , Polissacarídeos/biossíntese , Veias Umbilicais/metabolismo , Amino Açúcares/isolamento & purificação , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Feminino , Glicoproteínas/isolamento & purificação , Humanos , Meliteno/farmacologia , Polissacarídeos/isolamento & purificação , GravidezRESUMO
The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.
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Endotélio/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Fosfatidilcolinas/biossíntese , Animais , Bovinos , Células Cultivadas , Cromatografia em Camada Fina , Vasos Coronários/metabolismo , Histamina/farmacologia , Humanos , Hidrólise , Ácidos Hidroxieicosatetraenoicos/farmacologia , Metabolismo dos Lipídeos , Peptidil Dipeptidase A/metabolismo , Prostaglandinas/biossíntese , Cordão Umbilical/metabolismoRESUMO
Human umbilical vein endothelial cells were found to contain adrenic acid (22:4) in their cellular lipids. Since this fatty acid may be metabolized by cyclooxygenase in the kidney, the metabolism of adrenic acid was studied in endothelial cell cultures. [14C]Adrenic acid was metabolized to several more polar metabolites. Two of these metabolites co-migrated on HPLC with 1 alpha,1 beta-dihomo-8-ketoprostaglandin F1 alpha (the metabolite of 1 alpha, 1 beta-dihomoprostaglandin I2) and 1 alpha,1 beta-dihomoprostaglandin E2. Indomethacin (10(-5) M) inhibited the synthesis of these metabolites. When cells were treated with adrenic acid (3 X 10(-5) M), a peak that co-migrated with dihomo-8-ketoprostaglandin F1 alpha was detected by radioimmunoassay using an antiserum directed against 6-ketoprostaglandin F1 alpha. The presence of dihomo-8-ketoprostaglandin F1 alpha was confirmed by gas chromatography-mass spectrometry. Immunoreactive peaks that co-migrated with dihomoprostaglandins E2 and F2 alpha were identified with antisera against prostaglandin E2 and prostaglandin F2 alpha, respectively. [14C]Arachidonic acid was metabolized to [14C]prostaglandin F2 alpha, 6-keto[14C]prostaglandin F1 alpha, and [14C]prostaglandin E2. Similar results were found with unlabelled arachidonic acid using specific antisera. When the two fatty acids were combined, adrenic acid reduced the metabolism of arachidonic acid. The culture media from endothelial cells inhibited thrombin-induced platelet aggregation, an effect blocked by aspirin. The inhibitory activity of the media was enhanced when arachidonic acid was added to the cells, but it was reduced by adrenic acid. Both prostaglandin I2 and dihomoprostaglandin I2 inhibited platelet aggregation, but prostaglandin I2 was 100-times more potent. We conclude that adrenic acid is metabolized in human endothelial cells to 1 alpha, 1 beta-dihomoprostaglandins and can compete with endogenous arachidonic acid for conversion by cyclooxygenase. These findings suggest that adrenic acid may reduce the formation of prostaglandin I2 by the blood vessel.
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Dinoprostona/análogos & derivados , Endotélio/metabolismo , Ácidos Erúcicos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Meios de Cultura/farmacologia , Endotélio/efeitos dos fármacos , Epoprostenol/biossíntese , Epoprostenol/farmacologia , Ácidos Graxos/análise , Humanos , Indometacina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/análise , Prostaglandinas E/biossíntese , Prostaglandinas F Sintéticas/biossíntese , Trombina/farmacologia , Veias UmbilicaisRESUMO
Neutral endopeptidase 24.11 (NEP/CALLA/CD10), an enzyme expressed on early lymphoid progenitors, neutrophils, and various other cell types, inactivates many biologically active peptides, including the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP. Because PMNs in inflammatory sites are exposed to various cytokines, we evaluated the effects of selected cytokines on CD10/NEP activity in vitro. Of five cytokines tested--interleukin-1 (IL-1), IL-6, and IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF)--GM-CSF provided the most consistent increase in surface NEP activity. Low concentrations (10(-9)-10(-7) M) of GM-CSF increased NEP activity in a time- and concentration-dependent manner to more than 225% that of control (phosphate-buffered saline-treated) cells. Cytofluorometry of cells stained with a fluorescent antibody to CD10 indicated that GM-CSF increased expression of surface CD10/NEP antigen in a similar manner. The effect of GM-CSF on NEP activity was enhanced still further by simultaneous exposure to IL-1, suggesting that combinations of cytokines may direct and regulate the neutrophil response within an inflammatory site. Rapid upregulation of CD10/NEP underscores the importance of this enzyme for control of peptide mediators of inflammation.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Neprilisina/sangue , Neutrófilos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Interleucina-1/farmacologia , Neutrófilos/enzimologia , Proteínas Recombinantes/farmacologia , Regulação para CimaRESUMO
L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Cisteína/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Histidina/efeitos dos fármacos , Isoxazóis/farmacologia , Solventes/farmacologia , Sequência de Aminoácidos , Ácido Aspártico/efeitos dos fármacos , Ácido Glutâmico/efeitos dos fármacos , Cinética , Modelos Químicos , Dados de Sequência Molecular , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/análiseRESUMO
The vasodilator prostaglandins (PGs), prostacyclin (PGI2) and PGE2, may contribute to the inflammatory response. Because glucocorticosteroids reduce inflammation, possibly through inhibition of arachidonic acid release, we examined the influence of dexamethasone on PG formation in cultures of human endothelial cells. Binding of [3H]dexamethasone by intact cells was competed by unlabeled steroids and was half-maximal at 1.2 X 10(-8) M. A cytosolic fraction complexed with [3H]dexamethasone and migrated on sucrose density gradient centrifugation with a sedimentation coefficient of 8S. 3H-steroid binding was diminished by unlabeled steroid. Histamine, bradykinin, and the ionophore, A23187, stimulated release of PGI2 and PGE2 to as much as 25 times basal release. Dexamethasone (10(-11) to 10(-7) M) reduced PG formation in cells that were stimulated by histamine, bradykinin, calcium ionophore, or mechanical agitation. The inhibitory effect required at least 4 h to develop, was maximal at 24 h, and persisted after the steroid was removed. Hydrocortisone and triamcinolone had similar effects but were less potent than dexamethasone. Testosterone and progesterone did not affect PG generation. Both arachidonic acid and PGH2 augmented formation of PGs but were not inhibited by dexamethasone. Cortisol-21-mesylate, an antagonist of glucocorticoid receptors, blocked the effects of dexamethasone on PG formation, as did treatment of the cells with cycloheximide. We conclude that glucocorticoids inhibit PG production in endothelial cells by interaction with specific steroid receptors. The steroid-mediated inhibitory effect occurs at the level of arachidonic acid release and depends upon protein synthesis.