RESUMO
PURPOSE: Perforated peptic ulcer is one of the most common surgical emergencies worldwide. With the improvement in medical therapy for peptic ulcers, the number of elective surgical procedures has come down. However, the incidence of perforated peptic ulcer is still increasing and remains as a substantial health problem with significant postoperative morbidity and mortality. This study aimed to find out the association between various preoperative and intraoperative factors with the postoperative mortality and morbidity in patients operated for peptic ulcer perforation. METHODS: This prospective observational study had a time based sample of 101 perforation peritonitis cases admitted to the surgical wards of a tertiary care center from February 2015 to January 2016 who underwent laparotomy, diagnosed to have peptic ulcer perforation and underwent simple closure with an omental patch. Data regarding age, gender, presenting complaints, time elapsed from the onset of symptoms to surgery, physical examination findings, comorbid diseases, laboratory and imaging findings, intraoperative findings, length of hospital stay, postoperative morbidity, and mortality were recorded and analyzed. RESULTS: Female gender, older age group, perforation surgery interval more than 36 h, and size of perforation more than 1 cm2 were found to be significant factors influencing postoperative mortality and morbidity. Postoperative morbidity was also associated with comorbid diseases. Abnormal renal function on presentation was identified as an additional risk factor for postoperative morbidity and longer hospital stay. CONCLUSIONS: An understanding of these factors, identification of patients at risk and early intervention can help in reducing the postoperative morbidity and mortality in peptic ulcer perforation.
Assuntos
Úlcera Péptica Perfurada/mortalidade , Adulto , Feminino , Humanos , Incidência , Índia , Laparoscopia , Masculino , Pessoa de Meia-Idade , Úlcera Péptica Perfurada/cirurgia , Complicações Pós-Operatórias , Período Pré-Operatório , Estudos Prospectivos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
This article presents the development of a Portable Aerosol Collector and Spectrometer (PACS), an instrument designed to measure particle number, surface area, and mass concentrations continuously and time-weighted mass concentration by composition from 10 nm to 10 µm. The PACS consists of a six-stage particle size selector, a valve system, a water condensation particle counter to detect number concentrations, and a photometer to detect mass concentrations. The stages of the selector include three impactor and two diffusion stages, which resolve particles by size and collect particles for later chemical analysis. Particle penetration by size was measured through each stage to determine actual collection performance and account for particle losses. The data inversion algorithm uses an adaptive grid-search process with a constrained linear least-square solver to fit a tri-modal (ultrafine, fine, and coarse), log-normal distribution to the input data (number and mass concentration exiting each stage). The measured 50% cutoff diameter of each stage was similar to the design. The pressure drop of each stage was sufficiently low to permit its operation with portable air pumps. Sensitivity studies were conducted to explore the influence of unknown particle density (range from 500 to 3,000 kg/m3) and shape factor (range from 1.0 to 3.0) on algorithm output. Assuming standard density spheres, the aerosol size distributions fit well with a normalized mean bias of -4.9% to 3.5%, normalized mean error of 3.3% to 27.6%, and R2 values of 0.90 to 1.00. The fitted number and mass concentration biases were within ±10% regardless of uncertainties in density and shape. However, fitted surface area concentrations were more likely to be underestimated/overestimated due to the variation in particle density and shape. The PACS represents a novel way to simultaneously assess airborne aerosol composition and concentration by number, surface area, and mass over a wide size range.
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Hereditary Haemorrhagic Telangiectasia, also known as Osler-Rendu-Weber disease is a rare autosomal dominant disorder affecting small vessels of multiple systems whose main pathological change is the presence of abnormal arteriovenous communications. Usually presents as skin and mucosal telangiectasias, epistaxis, gastrointestinal bleeding and visceral arteriovenous malformations. Although the epistaxis and gastrointestinal blood loss can result in anaemia, patients with hereditary haemorrhagic telangiectasia rarely presents as severe anaemia1 or CNS infections. Herein, we report the case of a 57 year-old man who presented with severe anaemia resulting in congestive cardiac failure with history of recurrent blood transfusions and recurrent CNS infections which ultimately was diagnosed as hereditary haemorrhagic telangiectasia.
Assuntos
Telangiectasia Hemorrágica Hereditária/diagnóstico , Anemia/etiologia , Transfusão de Sangue , Infecções do Sistema Nervoso Central/etiologia , Epistaxe/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , RecidivaRESUMO
CONTEXT: There is enough documented evidence to prove the benefits of early and appropriate initiation of education among children with cerebral palsy (CP). AIM: To find out the proportion of children with CP who are enrolled for some kind of formal education and to study the determinants of the same. SETTING AND DESIGN: This cross sectional study was done among children, attending the special clinics at government medical college, Thiruvananthapuram. MATERIALS AND METHODS: Children between 3 and 12 years of age diagnosed with CP were subjects for the study. STATISTICAL ANALYSIS USED: Enrollment for any form of formal education was the major outcome variable. The factors associated with initiation of formal education were tested using Chi-square test or Fischer's exact test. Independent association of each factor was evaluated through binary logistic Regression analysis. RESULTS AND CONCLUSIONS: The mean (SD) age of the children (n = 86) was 5.7 (2.3) years with forty-six (53.5%) of them being girls. Diplegia was the commonest limb abnormality found. Fifty-two (60.5%) children were undergoing some kind of schooling. Those children who were less dependent physically and those who had achieved better language development were regular school goers. After binary logistic regression the ability of a child to speak in sentences (P = 0.008) and ambulatory level of the child (P = 0.019) were factors which favored, whereas delay in attaining the adaptive developmental milestone of transferring objects from one hand to another (P = 0.014) was found to be detrimental for school enrollment.
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Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of triglycerides, is primarily synthesized by adipocytes and myocytes. In addition to synthesis, degradation of cell surface-associated LPL is thought to be important in regulating production of the enzyme. We studied LPL metabolism in the LPL synthesizing adipocyte cell line BFC-1 beta and assessed the contributions of cell surface heparan sulfate proteoglycans (HSPG), low density lipoprotein receptor related protein (LRP), and glycosylphosphatidylinositol (GPI)-linked proteins to LPL uptake and degradation by these cells. Adipocytes degraded 10-12% of total cell surface I-labeled LPL in 2 h and 23-28% in 4 h. In 1 h, 30-54% of the degradation was inhibited by the 39 kDa receptor associated protein (RAP), an inhibitor of ligand binding to LRP. At 4 h, only 19-23% of the LPL degradation was RAP inhibitable. This suggested that two pathways with different kinetics were important for LPL degradation. Heparinase/heparitinase treatment of cells showed that most LPL degradation required the presence of HSPG. Treatment with phosphatidylinositol-specific phospholipase C (PIPLC) inhibited 125I-labeled LPL degradation by 13%. However, neither RAP nor PIPLC treatment of adipocytes significantly increased the amount of endogenously produced LPL activity in the media. To determine whether direct uptake of LPL bound to HSPG could account for the non-RAP sensitive LPL uptake and degradation, proteoglycan metabolism was assessed by labeling cells with 35SO4. Of the total pericellular proteoglycans, 14% were PIPLC releasable; surprisingly, 30% were dissociated from the cells with heparin. The amount of labeled pericellular proteoglycans decreased 26% in 2 h and 50% in 8 h, rapid enough to account for at least half of the degradation of cell surface LPL. We conclude that adipocytes degrade a fraction of the cell surface LPL, and that this process is mediated by both proteoglycans and RAP-sensitive receptors.
Assuntos
Tecido Adiposo/metabolismo , Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Lipase Lipoproteica/metabolismo , Chaperonas Moleculares/metabolismo , Proteoglicanas/metabolismo , Animais , Células CHO , Cricetinae , Heparina/metabolismo , Heparina Liase , Proteína Associada a Proteínas Relacionadas a Receptor de LDL , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/metabolismo , Polissacarídeo-Liases/metabolismoRESUMO
Previous studies (Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J.(1994) J. Biol. Chem. 269, 9409-9412) from this laboratory showed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL). LPL binding to endothelial cells, we hypothesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-surface NTAB would increase the amount and affinity of LPL binding to cells, we produced stable Chinese hamster ovary cell lines that have NTAB anchored to the cell surface. A cDNA encoding the amino-terminal 17% of apoB (apoB17) was fused to a cDNA coding for the last 37 amino acids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment. The fused construct was sequence-verified and cloned into expression vector pCMV5. The pCMV5-apoB17-DAF plasmid was cotransfected with a neomycin resistance gene into wild-type (WT) cells and mutant heparan sulfate proteoglycan-deficient Chinese hamster ovary cells (745 cells), and stable cell lines were established. Expression of apoB17 on the cell surface was confirmed by the release of apoB17 by phosphatidylinositol-specific phospholipase C. LPL binding to WT and apoB17-DAF-transfected cells was determined. Using 0.8-6 microg of LPL, 1.3-2.2-fold more LPL associated with apoB17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells. After heparinase treatment, LPL binding to apoB17-DAF cells was still greater than to treated WT cells. This increased binding to apoB17-DAF cells was almost abolished by treatment of cells with phosphatidylinositol-specific phospholipase C or anti-apoB monoclonal antibody. LPL dissociated from WT cells with k-1 = 2.55 x 10(-2) min-1, whereas LPL dissociated more slowly from apoB17-DAF-containing cells with k-1 = 1.08 x 10(-2) min-1. Furthermore, almost 95% of the LPL on WT cells was dissociated by 1 M NaCl, while only 65% of the LPL dissociated from apoB17-DAF cells at the same high salt concentration. Similarly, in high salt, more LPL remained associated with apoB17-DAF cells than with nontransfected 745 cells. These data show that NTAB on cell surfaces can function as a LPL-binding protein. Moreover, they demonstrate that LPL association with cells can be increased by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.
Assuntos
Apolipoproteínas B/metabolismo , Lipase Lipoproteica/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Apolipoproteínas B/química , Apolipoproteínas B/genética , Antígenos CD55/genética , Antígenos CD55/metabolismo , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetinae , Glicosilfosfatidilinositóis/metabolismo , Heparina/metabolismo , Heparina Liase , Hidrólise , Radioisótopos do Iodo , Fragmentos de Peptídeos/genética , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/metabolismo , Polissacarídeo-Liases/metabolismo , Ensaio Radioligante , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Cloreto de Sódio/farmacologia , Especificidade por Substrato , TransfecçãoRESUMO
We reported previously that a 116-kDa lipoprotein lipase (LPL)-binding protein from endothelial cells has sequence homology to the amino-terminal region of apolipoprotein (apo) B. We now tested whether endothelial cells synthesize apoB mRNA and protein. Primers were designed to the human apoB cDNA sequence and reverse transcription polymerase chain reaction was performed using total RNA isolated from bovine and human endothelial cells. With primers to the 5' region of the apoB mRNA (amino-terminal region of apoB protein) expected size PCR products were generated from both bovine and human endothelial cells as well as from mouse liver RNA, which was used as a control. Primers designed to the 3' region of apoB mRNA generated PCR products from human endothelial cells and HepG2 cells but not from bovine or mouse cells. These data suggest that endothelial cells contain full-length apoB mRNA and that the 5' or the amino-terminal region of apoB is highly conserved from mouse to human. This was confirmed by direct sequencing of the mouse and bovine PCR products. To test whether apoB protein was produced, bovine endothelial cell proteins were metabolically labeled with [35S]methionine/cysteine or [3H]leucine and immunoprecipitated with anti-human apoB antibodies. Using extracts from cells labeled for 1 h, monoclonal antibody 47, directed to the low density lipoprotein receptor binding region of apoB, precipitated a protein of approximate molecular mass 550,000, the size of full-length apoB. Immunoprecipitation of the 550-kDa protein was abolished in the presence of added unlabeled low density lipoprotein. From cells labeled for 16 h, a 116-kDa protein was immunoprecipitated by polyclonal anti-apoB antibodies. This protein was partly released from cells by heparin treatment. Pulse-chase analysis showed that the 116-kDa fragment appeared at the same time as the full-length apoB began disappearing. The immunoprecipitated 116-kDa fragment also bound labeled LPL on ligand blot, further suggesting that it is an amino-terminal fragment of apoB. Incubation of endothelial cells with oleic acid (0.25 and 0.5 mM) did not significantly alter the production of either the full-length apoB or the 116-kDa fragment. These data show that endothelial cells synthesize apoB. The full-length apoB appears to be cleaved to form a 116-kDa fragment that can function as a LPL-binding protein.
Assuntos
Apolipoproteínas B/biossíntese , Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Animais , Aorta , Apolipoproteínas B/química , Sequência de Bases , Carcinoma Hepatocelular , Bovinos , Linhagem Celular , Células Cultivadas , Sequência Conservada , Primers do DNA , DNA Complementar , Endotélio Vascular/efeitos dos fármacos , Biblioteca Gênica , Humanos , Intestinos , Cinética , Lipase Lipoproteica/metabolismo , Neoplasias Hepáticas , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Ácido Oleico , Ácidos Oleicos/farmacologia , Células Tumorais CultivadasRESUMO
Oligosaccharides (hexa to dodeca) terminating with [3H]2,5-anhydromannitol (AManR) were isolated from heparin by partial cleavage with nitrous acid at low pH (pH 1.5) followed by gel filtration and reduction with [3H]NaBH4. They were subsequently chromatographed on a lipoprotein lipase (LpL)-Sepharose column. High- and low-affinity oligosaccharides for LpL were isolated and characterized. Disaccharide analysis revealed the presence of (IdceA(2-SO4)-->AManR6-SO4) and (IdceA(2-SO4)-->AManR) as the major disaccharide products after low pH nitrous acid treatment. The oligosaccharides are, therefore, enriched in IdceA(2-SO4)-(GlcNSO4 +/- 6-SO4) sequences. Furthermore, they are found to be composed of 2-O-sulfated hexuronic acid-containing sequences, structural features, characteristic of heparin and heparan sulfate oligosaccharides with potential antiproliferative activities. These oligosaccharides may have the potential as lipase-releasing agents from endothelial and adipocyte surfaces.
Assuntos
Cromatografia de Afinidade/métodos , Heparina/química , Ácidos Hexurônicos/análise , Lipase Lipoproteica/metabolismo , Oligossacarídeos/isolamento & purificação , Boroidretos/metabolismo , Cromatografia em Agarose , Cromatografia em Camada Fina , Dissacarídeos/química , Dissacarídeos/isolamento & purificação , Hidrólise , Ácido Nitroso , Oligossacarídeos/química , SulfatosRESUMO
The cause and consequence of altered proteoglycans in atherosclerosis are poorly understood. To determine whether proteoglycans affect monocyte binding, we studied the effects of heparin and proteoglycan degrading enzymes on THP-1 monocyte adhesion to subendothelial matrix (SEM). Monocyte binding increased about 2-fold after SEM was treated with heparinase. In addition, heparin decreased monocyte binding to fibronectin, a known SEM protein, by 60%. These data suggest that SEM heparan sulfate inhibits monocyte binding to SEM proteins. We next examined whether lysolecithin, a constituent of modified lipoproteins, affects endothelial heparan sulfate proteoglycan (HSPG) production and monocyte binding. Lysolecithin (10-200 microM) decreased total 35SO4 in SEM (20-75%). 2-fold more monocytes bound to SEM from lysolecithin treated cells than to control SEM. Heparinase treatment did not further increase monocyte binding to lysolecithin-treated SEM. HSPG degrading activity was found in medium from lysolecithin-treated but not control cells. 35SO4-labeled products obtained from labeled matrix treated with lysolecithin-conditioned medium were similar in size to those generated by heparinase. These data suggest that lysolecithin-treated endothelial cells secrete a heparanase-like activity. We hypothesize that decreased vessel wall HSPG, as occurs in atherogenic conditions, allows increased monocyte retention within the vessel and is due to the actions of an endothelial heparanase.
Assuntos
Endotélio Vascular/fisiologia , Heparitina Sulfato/metabolismo , Lisofosfatidilcolinas/farmacologia , Monócitos/fisiologia , Proteoglicanas/metabolismo , Animais , Aorta , Autorradiografia/métodos , Bovinos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Condroitinases e Condroitina Liases/farmacologia , Meios de Cultivo Condicionados , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/efeitos dos fármacos , Proteoglicanas de Heparan Sulfato , Heparina Liase , Heparitina Sulfato/biossíntese , Heparitina Sulfato/isolamento & purificação , Humanos , Cinética , Lipoproteínas LDL/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Polissacarídeo-Liases/metabolismo , Polissacarídeo-Liases/farmacologia , Proteoglicanas/biossíntese , Proteoglicanas/isolamento & purificação , Sulfatos/metabolismoRESUMO
Lipoprotein lipase (LPL) hydrolyzes chylomicron and very low density lipoprotein (VLDL) triglycerides and potentiates the cellular uptake of lipoproteins. These LPL-lipoprotein associations could involve only protein-lipid interaction, or they could be modulated by apolipoproteins (apo). ApoB is the major protein component of chylomicrons, VLDL, and low density lipoprotein (LDL). ApoB100, a large glycoprotein with a molecular mass of 550 kDa, is composed of several functional domains. A carboxyl-terminal region of the protein is the ligand for the LDL receptor. There are several hydrophobic domains that are believed to be important in lipid binding. The relatively hydrophilic amino-terminal region of apoB, however, has no known function. Using solid phase assays we quantified LPL-lipoprotein complex formation. On a molar basis, severalfold greater amounts of LPL bound to LDL and VLDL than to high density lipoprotein at all the concentrations of LPL tested (0.9-55 nM). To assess the roles of LDL protein versus lipid, we performed competition and ligand blotting experiments. LDL and an amino-terminal fragment of apoB competed better for 125I-LPL binding to LDL than did lipid emulsion particles. Delipidation of LDL-coated plates did not alter LPL binding. On ligand blots, LPL bound to amino-terminal fragments of apoB generated by thrombin digestion but not to apoA1, apoE, or carboxyl-terminal fragments of apoB. Further evidence for LPL interaction with the amino-terminal region of apoB was obtained using anti-apoB monoclonal antibodies. Antibodies directed against the amino-terminal regions of apoB blocked LPL interaction with LDL, whereas those against the carboxyl-terminal region of apoB did not inhibit LPL interaction with LDL. Thus, we conclude that a specific interaction between LPL and the amino-terminal region of apoB may facilitate LPL association with circulating lipoproteins.
Assuntos
Apolipoproteínas B/metabolismo , Lipase Lipoproteica/metabolismo , Lipoproteínas/metabolismo , Anticorpos Monoclonais/farmacologia , Ligação Competitiva/efeitos dos fármacos , Humanos , Ligação ProteicaRESUMO
Lipoprotein lipase (LpL) catalyzes the hydrolysis of triglycerides in plasma lipoproteins at the luminal surface of the vascular endothelium. This enzyme is bound via electrostatic interactions to heparan sulfate (HS). The specific endothelial cell surface HS oligosaccharide sequences that are necessary for binding of LpL to HS have not been characterized. To identify this LpL-binding oligosaccharide sequence, oligosaccharides were isolated from bovine aortic endothelial cell-derived HS and assessed for LpL binding properties. Endothelial HS chains that were isolated from endothelial total cell-associated proteoglycans were deacetylated by complete hydrazinolysis, cleaved with nitrous acid (pH 4.5), and reduced with [3H]NaBH4. The resulting fragments composed of N-sulfated glucosamine-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) were chromatographed on a LpL-Sepharose column. A high affinity decasaccharide was isolated and characterized. Disaccharide analysis of this decasaccharide indicated that it yielded only the disaccharide IdceA(2-SO4)-->AManR(6-SO4) on treatment with nitrous acid at low pH. Therefore, the sequence of the LpL-binding decasaccharide is [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4)) alpha 1-4]4-IdceA(2-SO4) alpha 1-4AManR(6-SO4) and is distinct from those that bind antithrombin and basic fibroblast growth factor. Partial depolymerization of endothelial HS chains with hydrazine/high pH nitrous acid treatment gave rise to lipase-binding oligosaccharides larger than decasaccharide. However, further complete depolymerization of these oligosaccharides resulted in only a high affinity decasaccharide composed of repeating disaccharide units of [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4))]. These results indicate that the decasaccharide is the active fragment that binds to LpL with high affinity. Molecular modeling studies of the decasaccharide indicate that it presents a linear array of negatively charged sulfate groups that may adopt a favorable disposition to bind to peptide region(s) comprised of basic amino acid residues of LpL with high affinity.
Assuntos
Endotélio Vascular/metabolismo , Heparitina Sulfato/química , Lipase Lipoproteica/metabolismo , Oligossacarídeos/química , Proteoglicanas/química , Animais , Sequência de Carboidratos , Bovinos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Dissacarídeos/análise , Endotélio Vascular/citologia , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/metabolismo , Proteoglicanas/metabolismo , Especificidade por SubstratoRESUMO
Lipoprotein lipase (LPL), the principal enzyme which hydrolyzes triglycerides in circulating plasma lipoproteins, functions while bound to the luminal surface of endothelial cells. LPL is a heparin-binding protein and has been assumed to associate with endothelial cell heparan sulfate proteoglycans (HSPG). Recently, using ligand blotting and affinity chromatography we identified a 116-kDa heparin-releasable LPL-binding protein (hrp-116) from endothelial cells which was not a HSPG (Sivaram, P., Klein, M. G., and Goldberg, I. J. (1992) J. Biol. Chem. 267, 16517-16522). This suggested that, like a number of other heparin-binding proteins, LPL binding to cells also involves non-HSPG proteins. Using heparin-agarose affinity chromatography, a 116-kDa LPL-binding protein was purified from endothelial cell extracts. Microsequencing of peptides generated by Lys-C protease digestion revealed complete homology with four different regions in the NH2-terminal part of human apolipoprotein B (apoB). Western blots using anti-apoB monoclonal antibodies (mAb) that recognize the NH2-terminal region of apoB confirmed that a 116-kDa fragment of apoB was present on endothelial cell membranes. Further evidence that LPL associates with the NH2-terminal region of apoB was obtained by showing 1) that an NH2-terminal fragment of apoB obtained from apoB-transfected CHO cells bound LPL on ligand blots and 2) that NH2-terminal fragments of apoB generated by thrombin digestion of low density lipoprotein bind LPL. Evidence that the NH2-terminal region of apoB mediates LPL interaction with endothelial cells was obtained using monoclonal antibodies. mAb3 and mAb19, which recognize epitopes near the NH2 terminus of apoB, inhibited 125I-LPL binding to cells by 60-65%. In contrast, mAb47, which has determinants at the COOH-terminal end of apoB, inhibited LPL binding by only about 10%. The inhibitory effects of mAb3 and mAb19 were abolished following treatment of cells with heparin, which removes the 116-kDa LPL-binding protein. Furthermore, incubation of 125I-LPL in medium containing an NH2-terminal apoB fragment reduced LPL binding to cells. These data suggest that an NH2-terminal fragment of apoB that binds to endothelial surfaces facilitates LPL binding to cells.
Assuntos
Apolipoproteínas B/metabolismo , Endotélio Vascular/metabolismo , Lipase Lipoproteica/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Aorta , Apolipoproteínas B/farmacologia , Western Blotting , Células CHO , Bovinos , Células Cultivadas , Cromatografia de Afinidade , Cricetinae , Humanos , Peso Molecular , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Receptores de Superfície Celular/isolamento & purificação , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Lipoprotein lipase (LPL) is synthesized by adipocytes, associated with the cell surface, and released from the cells when they are treated with heparin. Release of LPL from the adipocyte is required for LPL to migrate to its physiological site of action on the luminal surface of capillary endothelial cells. To better understand this process, we studied the interaction of LPL with adipocyte cell membrane proteins. With the use of a ligand blot method, LPL specifically bound to a heparin-releasable, 116-kDa protein on mouse-derived brown fat adipose cell (BFC-1 beta) and rat adipocyte membranes. A 116-kDa cell surface protein was metabolically labeled with [35S]methionine and bound to LPL-Sepharose. This suggested that the LPL-binding protein was synthesized by the cells. When BFC-1 beta were treated with heparin to eliminate heparin-sensitive cell surface binding sites, LPL binding to the cells decreased and release of newly synthesized LPL activity increased. 125I-labeled LPL binding to control cells was reduced (> 70%) by a 50-fold excess of unlabeled LPL. The residual LPL binding to heparin-treated cells was, however, not decreased by the addition of unlabeled LPL. These data imply that specific adipocyte surface LPL binding involves heparin-sensitive sites. We hypothesize that the heparin-releasable, 116-kDa LPL-binding protein mediates specific LPL binding to adipocytes and that LPL activity within adipose tissue is regulated, in part, by the interaction of LPL with this binding protein.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Proteínas de Transporte/metabolismo , Heparina/farmacologia , Lipase Lipoproteica/metabolismo , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Eletroforese em Gel de Poliacrilamida , Epididimo , Feminino , Cinética , Lipase Lipoproteica/isolamento & purificação , Masculino , Metionina/metabolismo , Leite/enzimologia , Peso Molecular , Monensin/farmacologia , Ratos , Ratos Sprague-Dawley , Radioisótopos de EnxofreRESUMO
Lipoprotein lipase (LPL) hydrolyzes circulating lipoprotein triglyceride molecules while it is associated with the luminal surface of capillary endothelial cells. The precise molecular mechanism by which LPL attaches to these cells is unknown. LPL and a number of other molecules, including growth factors and clotting factors, bind to heparin-affinity gels and are eluted using high concentrations of salt. Of these molecules, antithrombin III and basic fibroblast growth factor have been shown to bind to specific cell surface heparan sulfate proteoglycans. Recent data from our laboratory (Sivaram et al. 1992. J. Biol. Chem. 267: 16517-16522) have shown that a heparin-sensitive, non-proteoglycan 116-kDa LPL-binding protein is present on cultured bovine aortic endothelial cells (BAEC). A series of experiments was performed to study the specificity of LPL binding to BAEC and to this 116-kDa protein. At low amounts of LPL (1 microgram) 125I-labeled LPL binding to the cells was inhibited up to 82% by the addition of a 20-fold excess of unlabeled LPL. LPL binding to the BAEC was not decreased by the addition of similar amounts of either antithrombin or thrombin. Specific LPL binding was eliminated by incubating the BAEC at 4 degrees C with heparin containing buffer prior to the addition of LPL. Although cellular internalization of 125I-labeled LPL at 37 degrees C was decreased when an excess of each of the three proteins was added to the culture medium, LPL was most effective. Furthermore, when LPL interaction with the 116-kDa binding protein was studied using ligand blots, 125I-labeled LPL binding was blocked only by unlabeled LPL.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Endotélio Vascular/enzimologia , Lipase Lipoproteica/metabolismo , Animais , Antitrombina III/metabolismo , Antitrombinas/farmacologia , Aorta/enzimologia , Sítios de Ligação , Ligação Competitiva , Bovinos , Células Cultivadas , Cães , Endotélio Vascular/efeitos dos fármacos , Heparina/farmacologia , Lipase Lipoproteica/farmacologia , Peso Molecular , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Trombina/farmacologiaRESUMO
Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HL) were biotinylated using N-hydroxysuccinamide ester of biotin (25-fold molar excess) which was incorporated into the lysine amino groups of the enzyme protein. By assessing enzyme activity and heparin-agarose affinity a biotinylation protocol which did not denature lipases was developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that biotinylated LPL (bLPL) has the same mobility as that of unlabeled or iodinated LPL. Receptor binding activity of bLPL was studied in (i) cell binding experiments using cultured bovine aortic endothelial cells and (ii) ligand blotting experiments using endothelial cell plasma membranes. Endothelial cells in culture bound similar amounts of bLPL and 125I-LPL. We previously described a 116-kDa heparin-releasable LPL binding protein (hrp-116) on endothelial cells. Using biotinylated lipases in ligand blotting experiments we now demonstrate that both bLPL and biotinylated HL can bind to hrp-116. bLPL in addition also bound to low-density lipoprotein receptor related protein in ligand blotting. Thus, our protocol has produced biotinylated lipases which are both chemically and biologically active and can be used instead of iodinated lipases.
Assuntos
Biotina , Lipase/química , Lipase Lipoproteica/química , Fígado/enzimologia , Receptores de Superfície Celular/análise , Animais , Bovinos , Células Cultivadas , Cromatografia em Agarose , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Immunoblotting , LigantesRESUMO
Triglycerides in circulating plasma lipoproteins are hydrolyzed by lipoprotein lipase (LPL) which is thought to bind to proteoglycans on the luminal endothelial cell surface. Previous studies from this laboratory using LPL-Sepharose affinity chromatography identified a 220-kDa LPL binding proteoglycan. Using ligand blotting with 125I-LPL, we now report a 116-kDa LPL binding protein in plasma membrane preparations of endothelial cells. 125I-LPL binding to this protein was abolished by addition of unlabeled LPL. When the cell surface of endothelial cells was labeled with biotin, a 116-kDa protein was biotinylated. Furthermore, the biotinylated 116-kDa protein bound to LPL-Sepharose and eluted with 0.4 M NaCl suggesting that the 116-kDa LPL binding protein is present on the cell surface. When detergent extracts of endothelial cells were applied to LPL-Sepharose in the presence of 0.15 M NaCl, the 116-kDa, but not the 220-kDa, protein still bound to LPL-Sepharose. The 116-kDa protein was not labeled with 35SO4 and eluted from DEAE-cellulose prior to proteoglycans, suggesting that it is not a proteoglycan. However, a 116-kDa endothelial cell surface protein was metabolically labeled with [35S]methionine. This protein was dissociated from the cell surface by incubating cells with heparin (50 units/ml)-containing buffer. After heparin treatment of endothelial cells, LPL binding to and internalization by the cells decreased greater than 70% compared to control cells. These results suggest that endothelial cells synthesize a heparin-releasable, high affinity 116-kDa LPL binding protein. We postulate that this protein is associated with proteoglycans on luminal endothelial surfaces and mediates LPL binding, internalization, and recycling. We name this protein hrp (heparin-releasable protein)-116.
Assuntos
Proteínas de Transporte/metabolismo , Endotélio Vascular/metabolismo , Heparina/farmacologia , Lipase Lipoproteica/metabolismo , Proteínas de Membrana/metabolismo , Animais , Aorta , Proteínas de Transporte/isolamento & purificação , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Proteínas de Membrana/isolamento & purificação , Peso MolecularRESUMO
Arginyl-tRNA synthetase (arginine-tRNA ligase, EC 6.1.1.19) is found in extracts of mammalian cells both as a free protein (Mr = 60,000) and as a component (Mr approximately 72,000) of the high molecular weight aminoacyl-tRNA synthetase complex (Mr greater than 10(6). Several pieces of evidence indicate that the low molecular weight free form is not a proteolytic degradation product of the complex-bound enzyme but that it preexists in vivo: (i) the endogenous free form differs in size from the active proteolytic fragment generated in vitro, (ii) conditions expected to increase or decrease the amount of proteolysis do not alter the ratio of the two forms of the enzyme, and (iii) the free form contains an NH2-terminal methionine residue. A model is presented that provides a rationale for the existence of two forms of arginyl-tRNA synthetase in cells. In this model the complexed enzyme supplies arginyl-tRNA for protein synthesis, whereas the free enzyme provides arginyl-tRNA for the NH2-terminal arginine modification of proteins by arginyl-tRNA:protein arginyltransferase. This latter process targets certain proteins for removal by the ubiquitin-dependent protein degradation pathway. The necessity for an additional pool of arginyl-tRNA for the modification reaction leads to the conclusion that the arginyl-tRNA destined for protein synthesis (and/or protein modification) is channeled and unavailable for other processes. Other evidence supporting channeling in protein synthesis is discussed.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Arginina-tRNA Ligase/metabolismo , Isoenzimas/metabolismo , Fígado/enzimologia , Biossíntese de Proteínas , Aminoacil-RNA de Transferência/metabolismo , Sequência de Aminoácidos , Animais , Arginina-tRNA Ligase/isolamento & purificação , Cromatografia em Gel , Escherichia coli/enzimologia , Isoenzimas/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
Aminoacyl-tRNA synthetases from higher eukaryotes often are isolated as high molecular weight complexes associated with other components such as lipids. Since hydrophobic interactions are involved in the organization of the complex, it has been suggested that interaction of synthetases with these lipids might be important for their structure and function. Delipidation is known to affect certain properties of synthetases within the complex including sensitivity to detergents plus salts, temperature inactivation, hydrophobicity, sensitivity to proteases, and, as shown here, sensitivity to p-mercuribenzoate and sites of papain cleavage. Of the lipids known to co-purify with the complex, cholesterol esters, phospholipids and free fatty acids, we show that the particular lipids responsible for many of these changes are the free fatty acids. Specific removal of fatty acids results in a complex with properties similar to one totally delipidated by detergent treatment, and readdition of the fatty acid fraction reverses the effects. The fatty acid fraction contains both saturated and unsaturated fatty acids, but unsaturated fatty acids are much more effective in reversing the properties of the delipidated complex that are saturated fatty acids. These results indicate that the free fatty acids co-purifying with the synthetase complex bind to the synthetases and affect their structure and function.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Aminoacil-tRNA Sintetases/análise , Animais , Arginina-tRNA Ligase/metabolismo , Western Blotting , Fenômenos Químicos , Físico-Química , Cromatografia , Detergentes/farmacologia , Ácidos Graxos/farmacologia , Ácidos Graxos não Esterificados/análise , Ácidos Graxos não Esterificados/farmacologia , Feminino , Mercurobenzoatos/farmacologia , Peso Molecular , Papaína/metabolismo , Coelhos , Ratos , Ratos Endogâmicos , Relação Estrutura-AtividadeRESUMO
The high molecular weight aminoacyl-tRNA synthetase complexes found in extracts of many eukaryotic cells often contain lipids and other non-protein components. Since hydrophobic interactions play an important role in maintaining synthetases in the complex, it has been suggested that the lipids present may also participate in its functional and structural integrity. In order to learn more about the role of lipids in the complex, we have compared the properties of the normal complex to one which has been delipidated by treatment with Triton X-114. Delipidation does not affect the size or activity of the aminoacyl-tRNA synthetase complex, but a variety of functional and structural properties of individual synthetases in the complex are altered dramatically. These include sensitivity to salts plus detergents, temperature inactivation, hydrophobicity, and sensitivity to protease digestion. In the latter case, removal of lipids also affects the low molecular weight products released by protease digestion. Purification of the synthetase complex by various chromatographic procedures can remove the lipids and lead to a structure that behaves like the delipidated complex prepared by detergent treatment. The significance of these findings for the intracellular location of aminoacyl-tRNA synthetases and for the study of purified complexes are discussed.