RESUMEN
BACKGROUND: Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. METHODS: An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. RESULTS: At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. CONCLUSION: These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.
Asunto(s)
Efrina-B2/química , Calicreínas/química , Calicreínas/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Calicreínas/fisiología , Masculino , Ratones , Datos de Secuencia Molecular , Trasplante de Neoplasias , Neoplasias de la Próstata , Proteolisis , Células Sf9RESUMEN
Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10(-22)). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10(-34)). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.
Asunto(s)
Predisposición Genética a la Enfermedad , Calicreínas/genética , Neoplasias de la Próstata/genética , ARN Mensajero/genética , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Simulación de Dinámica Molecular , Polimorfismo de Nucleótido Simple , Antígeno Prostático Específico/sangreRESUMEN
Pulmonary surfactant has been looked for and found in 11 representatives of four vertebrate classes. The amount of surfactant, estimated by quantitative spreading as a surface film, correlates well with alveolar surface area and with the amount of saturated, mainly dipalmitoyl, phosphatidylcholine in the lung parenchyma. The quantities of other phospholipids do not correlate well with alveolar surface area.
Asunto(s)
Lipoproteínas/análisis , Pulmón/fisiología , Fosfolípidos/análisis , Alveolos Pulmonares/análisis , Propiedades de Superficie , Tensoactivos/análisis , Animales , Anuros , Evolución Biológica , Caniformia , Bovinos , Pollos , Perros , Cobayas , Ratones , Fosfatidilcolinas/análisis , Conejos , Ratas , TortugasRESUMEN
This new enzymatic method disperses rabbit lung into morphologically intact, viable individual cells. The scattered cells constitute more than 50 percent of the original tissue. At least 90 percent of the cells exclude trypan blue from the nucleus. The dispersed lung cells consumed 6.2 microliters of oxygen per hour per milligram, dry weight. They incorporated [1-(14)C]palmitate into lecithin.
Asunto(s)
Pulmón/metabolismo , Animales , Isótopos de Carbono , Catalasa/metabolismo , Células Cultivadas , Desoxirribonucleasas/metabolismo , Pulmón/citología , Métodos , Colagenasa Microbiana/metabolismo , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Consumo de Oxígeno , Ácidos Palmíticos/metabolismo , Elastasa Pancreática/metabolismo , Papaína/metabolismo , Fosfatidilcolinas/biosíntesis , Pronasa/metabolismo , Conejos , Coloración y Etiquetado , Azul de TripanoRESUMEN
It is routine to genetically modify cells to express fluorescent or bioluminescent reporter proteins to enable tracking or quantification of cells in vitro and in vivo. Herein, we characterized the stability of luciferase reporter systems in C4-2B prostate cancer cells in mono-culture and in co-culture with bone marrow-derived mesenchymal stem/stromal cells (BMSC). An assumption made when employing the luciferase reporter is that the luciferase expressing cell number and bioluminescence signal are linearly proportional. We observed instances where luciferase expression was significantly upregulated in C4-2B cell populations when co-cultured with BMSC, resulting in a significant disconnect between bioluminescence signal and cell number. We subsequently characterized luciferase reporter stability in a second C4-2B reporter cell line, and six other cancer cell lines. All but the single C4-2B reporter cell population had stable luciferase reporter expression in mono-culture and BMSC co-culture. Whole-genome sequencing revealed that relative number of luciferase gene insertions per genome in the unstable C4-2B reporter cell population was lesser than stable C4-2B, PC3 and MD-MBA-231 luciferase reporter cell lines. We reasoned that the low luciferase gene copy number and genome insertion locations likely contributed to the reporter gene expression being exquisitely sensitive BMSC paracrine signals. In this study, we show that it is possible to generate a range of stable and reliable luciferase reporter prostate- and breast- cancer cell populations but advise not to assume stability across different culture conditions. Reporter stability should be validated, on a case-by-case basis, for each cell line and culture condition.
Asunto(s)
Luciferasas/aislamiento & purificación , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/aislamiento & purificación , Células Madre Mesenquimatosas/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica/genética , Genes Reporteros/genética , Humanos , Luciferasas/química , Proteínas Luminiscentes/química , Masculino , Células Madre Mesenquimatosas/patología , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Transgenes/genéticaRESUMEN
Bone metastases frequently occur in the advanced stages of breast cancer. At this stage, the disease is deemed incurable. To date, the mechanisms of breast cancer-related metastasis to bone are poorly understood. This may be attributed to the lack of appropriate animal models to investigate the complex cancer cell-bone interactions. In this study, two established tissue-engineered bone constructs (TEBCs) were applied to a breast cancer-related metastasis model. A cylindrical medical-grade polycaprolactone-tricalcium phosphate scaffold produced by fused deposition modelling (scaffold 1) was compared with a tubular calcium phosphate-coated polycaprolactone scaffold fabricated by solution electrospinning (scaffold 2) for their potential to generate ectopic humanised bone in NOD/SCID mice. While scaffold 1 was found not suitable to generate a sufficient amount of ectopic bone tissue due to poor ectopic integration, scaffold 2 showed excellent integration into the host tissue, leading to bone formation. To mimic breast cancer cell colonisation to the bone, MDA-MB-231, SUM1315, and MDA-MB-231BO breast cancer cells were cultured in polyethylene glycol-based hydrogels and implanted adjacent to the TEBCs. Histological analysis indicated that the breast cancer cells induced an osteoclastic reaction in the TEBCs, demonstrating analogies to breast cancer-related bone metastasis seen in patients.
Asunto(s)
Neoplasias Óseas/secundario , Huesos/patología , Neoplasias de la Mama/patología , Modelos Biológicos , Ingeniería de Tejidos/métodos , Animales , Neoplasias Óseas/patología , Calcificación Fisiológica/efectos de los fármacos , Fosfatos de Calcio/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Ratones SCID , Invasividad Neoplásica , Tamaño de los Órganos/efectos de los fármacos , Poliésteres/farmacología , Especificidad de la Especie , Andamios del Tejido/química , Microtomografía por Rayos XRESUMEN
Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Esferoides Celulares , Taxoides/farmacología , Células Tumorales CultivadasRESUMEN
We have previously demonstrated low levels of immunoreactive (ir)-beta-endorphin (beta-EP) and ir-ACTH in a subpopulation of mouse spleen macrophages, which is consistent with an involvement of opioid peptides in modulation of immune responses. Gel chromatography studies suggested the presence of an approximately 3.5,000-molecular weight (mol wt) species, putatively beta-EP, an approximately 11.5,000-mol-wt species, putatively beta-lipotropin, and a higher molecular weight species (putative beta-EP precursor, pro-opiomelanocortin (POMC). In this study we have extended our original findings by demonstrating the presence of messenger RNA for POMC by the use of a complementary DNA probe and Northern blot analysis of extracts of mouse and rat spleen. In addition, using high performance liquid chromatography (HPLC), we have shown that the major endorphin species in mouse spleen macrophages is beta-EP1-31, and that there are smaller amounts of each of the acetylated forms, N-acetyl-beta-EP1-16 (alpha-endorphin), N-acetyl-beta-EP1-17 (gamma-endorphin), N-acetyl-beta-EP1-27, and N-acetyl-beta-EP1-31. We interpret these studies as showing that (a) the spleen is an organ of POMC synthesis and that (b) the predominant COOH-terminal product of macrophage POMC is the opiate-receptor active species beta-EP1-31.
Asunto(s)
Endorfinas/biosíntesis , Macrófagos/metabolismo , Proopiomelanocortina/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Bazo/citología , Animales , Cromatografía Líquida de Alta Presión , ADN/análisis , Masculino , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , Poli A/metabolismo , ARN/metabolismo , betaendorfinaRESUMEN
In previous reports modest levels of beta-endorphin have been found by radioimmunoassay in rat testis, and localized by immunofluorescence to the interstitial cells. We have confirmed these previous reports and extended them by showing that the majority of testicular endorphins are acetylated forms, N-acetyl gamma-endorphin, N-acetyl alpha-endorphin, and N-acetyl beta-endorphin1-27. In addition, N-acetylated endorphins are not found in interstitial cells, but are confined to spermatogonia and primary spermatocytes.
Asunto(s)
Endorfinas/análisis , Espermatocitos/análisis , Espermatogonias/análisis , Espermatozoides/análisis , Envejecimiento , Animales , Técnica del Anticuerpo Fluorescente , Células Intersticiales del Testículo/análisis , Células Intersticiales del Testículo/fisiología , Masculino , Ratas , Ratas Endogámicas , Espermatocitos/fisiología , Espermatogonias/fisiología , betaendorfinaRESUMEN
The PROtein TOpography and Migration Analysis Platform (PROTOMAP) approach is a degradomics technique used to determine protease substrates within complex protein backgrounds. The method involves protein separation according to protein relative mobility, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel lanes are then sliced into horizontal sections, and in-gel trypsin digestion performed for each gel slice. Extracted peptides and corresponding proteins are identified using liquid chromatography-tandem mass spectrometry and bioinformatics. Results are compiled in silico to generate a peptograph for every identified protein, being a pictorial representation of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins shown by their peptograph to have migrated further through the gel (i.e., to a lower gel slice) in the lane containing the active protease(s) of interest, as compared to the control, are deemed putative protease substrates. PROTOMAP has broad applicability to a range of experimental conditions and protein pools. Coupling this with its simple and robust methodology, the PROTOMAP approach has emerged as a valuable tool with which to determine protease substrates in complex systems.
Asunto(s)
Péptido Hidrolasas/metabolismo , Péptidos , Proteínas/metabolismo , Proteómica/métodos , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Péptidos/química , Péptidos/metabolismo , Proteínas/química , Proteolisis , Programas Informáticos , Estadística como Asunto/métodos , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Flujo de TrabajoRESUMEN
Cancer is the second leading cause of death with 14 million new cases and 8.2 million cancer-related deaths worldwide in 2012. Despite the progress made in cancer therapies, neoplastic diseases are still a major therapeutic challenge notably because of intra- and inter-malignant tumour heterogeneity and adaptation/escape of malignant cells to/from treatment. New targeted therapies need to be developed to improve our medical arsenal and counter-act cancer progression. Human kallikrein-related peptidases (KLKs) are secreted serine peptidases which are aberrantly expressed in many cancers and have great potential in developing targeted therapies. The potential of KLKs as cancer biomarkers is well established since the demonstration of the association between KLK3/PSA (prostate specific antigen) levels and prostate cancer progression. In addition, a constantly increasing number of in vitro and in vivo studies demonstrate the functional involvement of KLKs in cancer-related processes. These peptidases are now considered key players in the regulation of cancer cell growth, migration, invasion, chemo-resistance, and importantly, in mediating interactions between cancer cells and other cell populations found in the tumour microenvironment to facilitate cancer progression. These functional roles of KLKs in a cancer context further highlight their potential in designing new anti-cancer approaches. In this review, we comprehensively review the biochemical features of KLKs, their functional roles in carcinogenesis, followed by the latest developments and the successful utility of KLK-based therapeutics in counteracting cancer progression.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Calicreínas/metabolismo , Neoplasias/enzimología , Péptido Hidrolasas/metabolismo , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Progresión de la Enfermedad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Calicreínas/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Péptido Hidrolasas/genética , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genéticaRESUMEN
Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.
Asunto(s)
Cadherinas/metabolismo , Calicreínas/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología , División Celular , Línea Celular Tumoral , Movimiento Celular , Células Epiteliales/patología , Humanos , Calicreínas/genética , Masculino , Mesodermo/patología , Invasividad NeoplásicaRESUMEN
The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.
Asunto(s)
Núcleo Celular/química , Citoplasma/química , Calicreínas/análisis , Neoplasias de la Próstata/metabolismo , Andrógenos/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Glicosilación , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Neoplasias de la Próstata/química , Neoplasias de la Próstata/genética , Biosíntesis de Proteínas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Semen/metabolismoRESUMEN
Surfactant phospholipid exists in multicompartment pools within the subphase of the lung. Movement among these pools and back into type II alveolar cells may be catalyzed by a phospholipid transfer protein resident in the subphase. We demonstrate here that a protein fraction obtained from canine lung lavage catalyzes the intermembrane transfer of all the major surfactant phospholipids. The protein is probably not derived from serum and is unrelated to surfactant proteins that have already been described.
Asunto(s)
Líquido del Lavado Bronquioalveolar/análisis , Fosfolípidos/metabolismo , Animales , Cromatografía en Gel , Perros , Masculino , Surfactantes Pulmonares/metabolismoRESUMEN
To estimate the turnover time of alveolar surfactant in New Zealand white rabbits, we injected [9,10-(3)H] palmitic acid and [2-(14)C] glycerol intravenously. From 1-48 h after injection, wer killed the animals, lavaged the lungs for alveolar surfactant with saline, and isolated the lamellar bodies by homogenization and sucrose density gradient centrifugation. Lamellar bodies and alveola surfactant had comparable phospholipid composition and surface activity. Lamellar bodies contained little DNA, no mitochondrial enzyme activity and less than 5% contaminating phospholipids from microsomal and Golgi-enriched fractions. We measured radioactivity of phosphatidylcholine, saturated phosphatidylcholine and phosphatidylglycerol for each isotope in lamellar bodies and surfactant at each time point. The plot of the integral with respect to time of the difference between lamellar body and surfactant specific activity against surfactant specific activity has a slope determined by the turnover time, and a shape which tests the precision of the precursor-product relationship. This analysis does not assume a pulse label and allows for possible recycling of tracer from surfactant to lamellar bodies. We obtained turnover times of 4-11 h. We detected an imprecise precursor-product relationship between lamellar bodies and alveolar surfactant, which is not due to experimental variability or to contamination of lamellar bodies by other subcellular fractions but may reflect imperfect mixing within surfactant compartments.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Fosfolípidos/metabolismo , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Radioisótopos de Carbono , Gránulos Citoplasmáticos/ultraestructura , Cinética , Masculino , Microscopía Electrónica , Alveolos Pulmonares/ultraestructura , Conejos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , TritioRESUMEN
The lung surfactant isolated from pulmonary fluid of fetal sheep changes both in amount and composition during gestation. Total phosphatidylcholine (PC) and its most surface-active components, disaturated PC, are present at very low levels 3-4 weeks prior to term and rise to adult levels 3-4 days before birth. The acidic phospholipids appear with a different time course. Phosphatidylserine reaches elevated levels about 21 days before birth. Phosphatidylinositol begins to increase at about 130 days of gestation. Phosphatidylglycerol is not a component (less than 1%) of the surfactant in this fetal lung fluid. At term, phosphatidylinositol is the major acidic phospholipid found in these fluids.
Asunto(s)
Pulmón/embriología , Fosfolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Fosfatasa Ácida/aislamiento & purificación , Animales , Fenómenos Químicos , Química , Femenino , Feto/metabolismo , Edad Gestacional , Pulmón/enzimología , Pulmón/metabolismo , Embarazo , Ovinos/metabolismoRESUMEN
The goal of this investigation was to characterize the proteins in subfractions of alveolar surfactant obtained by lung lavage and separated by differential centrifugation. It was previously demonstrated that the material in the more sedimentable fraction, which was enriched in tubular-myelin and was surface-active may be a precursor to the less sedimentable, vesicular, inactive material [1]. Separation of the proteins by polyacrylamide gel electrophoresis showed that the more sedimentable subfractions and rabbit surfactant isolated by conventional methods contained proteins with molecular weights comparable to those previously reported for alveolar surface active material (approximately 36 000 and 10 000). The less sedimentable subfractions contained less of these proteins. Immunoblots with anti-dog surfactant apoprotein antibodies, which cross-react with rabbit proteins, supported these observations. Immunoblots also showed that all of the subfractions contained serum proteins and secretory IgA, with the less sedimentable subfractions containing more secretory IgA. These results suggested that changes in protein composition may accompany functional changes in surfactant in the alveoli.
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Proteínas/aislamiento & purificación , Alveolos Pulmonares/análisis , Surfactantes Pulmonares/análisis , Animales , Electroforesis en Gel de Poliacrilamida , Membrana Dobles de Lípidos , Masculino , Microscopía Electrónica , Peso Molecular , Fosfolípidos/análisis , Conejos , Irrigación TerapéuticaRESUMEN
Surfactant protein C (SP-C) is a small hydrophobic protein that is synthesized and secreted by alveolar type II cells. The mechanism of clearance of SP-C from the alveolar airspace is not well understood, although previous studies demonstrated that recombinant SP-C instilled into the lungs of spontaneously breathing anaesthetized rats was taken up by type II cells and incorporated into lamellar bodies. The current investigation was undertaken to characterize the interaction of a complex of SP-C and surfactant-like lipids with freshly isolated rat alveolar type II cells under conditions in which the extracellular milieu can be regulated. SP-C was isolated from alveolar proteinosis lavage fluid and radiolabeled with 125I-Bolton-Hunter reagent. The radiolabeled protein retained its ability to facilitate adsorption of phospholipids to an air/liquid interface. Labeled human SP-C associated with isolated type II cells in a concentration-dependent manner that was also dependent upon temperature and time. The association of labeled SP-C with isolated type II cells did not saturate up to 150 micrograms/ml. SP-A significantly enhanced the association of SP-C with isolated type II cells. Under the experimental conditions tested, SP-C was not degraded to TCA-soluble products. These results are consistent with the hypothesis that association or uptake of SP-C by type II cells may be enhanced by SP-A and that like SP-A, SP-C is recycled by type II cells.
Asunto(s)
Macrófagos Alveolares/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Radioisótopos de Yodo , Masculino , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Ratas , Ratas Sprague-DawleyRESUMEN
Because previous studies have suggested that lung surfactant is not a simple compartment of homogeneous material, we subfractionated lamellar bodies and components of alveolar lavage from male New Zealand white rabbits, according to differences in sedimentability. We recovered two lamellar body populations at different densities in discontinuous sucrose density gradients; we separated six subfractions of alveolar lavage by differential centrifugation. To determine whether or not precursor-product relationships existed among the subfractions, we injected radioactive palmitate intravenously, killed the rabbits 1-72 h later, and measured phospholipid specific activities. The two populations of lamellar bodies had similar phospholipid composition, fatty acyl composition of phosphatidylcholine and phosphatidylglycerol, and surface activity. Light lamellar bodies had a higher ratio of phospholipid to protein, and labelled with tracer later in time than dense ones. For alveolar lavage subfractions, later labelling with tracer, lower adsorption rate and lower total protein and phosphatidylglycerol content seemed to correlate with decreasing average density and particle size as well as with the disappearance of tubular myelin structure and appearance of predominantly vesicular structure. The subfractions appear to be in a metabolic sequence in which heavier, more dense material is a precursor to lighter, less dense material. The results suggest that subfractions of surfactant are extensively recycled.