RESUMO
OBJECTIVES: To perform a synonymous, non-synonymous codon mutational analysis of the IGFBP gene family and identify mechanisms by which the IGFBP subfamilies diverged. METHODS: We identified 78 intact nucleotide sequences from 6 IGFBP subfamilies and 12 different species and used them for phylogenetic and synonymous, non-synonymous codon mutational analysis. Deletion and insertion comparisons were performed across subfamilies to determine if this might play a unique role in subfamily genesis. RESULTS: IGFBP-2 was identified by phylogenetic analysis to be the most related subfamily of the IGFBP progenitor, followed by IGFBP-4. Insertions and deletions within the variable domains were associated with divergence of each subfamily from its progenitor, suggesting a common motif for IGFBP evolution. Insertions unique to mammals were also found within the amino terminus of IGFBP-2. CONCLUSION: IGFBP subfamily divergence is associated with variable domain insertion or deletion and vigorous non-synonymous codon mutation. Our findings suggest strong selective pressure for IGFBP divergence in terrestrial vertebrates.
Assuntos
Evolução Molecular , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Sequência de Aminoácidos , Animais , Deleção de Genes , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/química , Dados de Sequência Molecular , Mutagênese Insercional , Mutação de Sentido Incorreto , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVES: To determine if plasmin differentially augments platelet aggregation through variable efficiencies of IGF-IGFBP complex cleavage. METHODS: We utilized ADP-triggered platelet aggregation assays to test the effects of IGF-I versus IGF-II in complex with IGFBP-2 or IGFBP-3 upon the efficiency of plasmin (a known IGFBP protease) as a pro-aggregatory stimulus. In vitro proteolysis assays were performed as controls. RESULTS: We found that IGF-I complexes augmented platelet-mediated aggregation whereas IGF-II either had no effect (IGFBP-2) or inhibited platelet-mediated aggregation (IGFBP-3). In vitro proteolysis assays of IGFBP-2 and IGFBP-3 using plasmin revealed that three of the four aggregation findings were explained by the disparate efficiencies of IGFBP proteolysis associated with each IGF. Only IGF-II-IGFBP-2 complex resulted in a finding that could not be explained by the concept of differential regulation of plasmin's proteolysis efficiency by the two IGF ligands. CONCLUSIONS: Our findings demonstrate that the plasmin can differentially modulate platelet aggregation in response to intrinsic heterogeneities within the IGF axis.
Assuntos
Fibrinolisina/fisiologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Agregação Plaquetária/fisiologia , Processamento de Proteína Pós-Traducional , Somatomedinas/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Fibrinolisina/metabolismo , Humanos , Indometacina/farmacologia , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Ligação ProteicaRESUMO
BACKGROUND: Early postnatal steroids and indomethacin in combination have been shown to increase the risk of spontaneous intestinal perforation (SIP) in infants with extremely low birth weight (ELBW), but the mechanism behind this synergistic effect is unknown. MATERIALS AND METHODS: Based on literature in a variety of models suggesting that glucocorticoids and nonsteroidal antiinflammatory agents diminish complementary isoforms of nitric oxide synthase (NOS), we hypothesized that perturbations in NO metabolism contribute to SIP. RESULTS: Our results using newborn wild-type (WT) and endothelial NOS-knockout (eNOS KO) mice treated with dexamethasone and/or indomethacin indicate that indomethacin treatment diminishes ileal eNOS abundance; dexamethasone treatment diminishes ileal inducible NOS and neuronal NOS (nNOS); 100% of dexamethasone-treated eNOS KO mice die after 3 days; eNOS KO mice treated for 2 days with dexamethasone develop acute pyloric stenosis in association with reduced expression of pyloric nNOS; and isolated ileum from eNOS KO mice treated for 2 days with dexamethasone exhibit a significant decrease in spontaneous peristalsis, decreased circumference, and decreased capacitance for forced volume before ileal perforation compared with ileum from untreated controls. CONCLUSIONS: Our findings suggest that eNOS and nNOS display functional overlap in the newborn mouse gastrointestinal tract and that simultaneous reduction in the activity of both NOS isoforms may be a risk factor for neonatal ileal perforation. If this holds true in human infants, then it provides a plausible etiologic explanation for the strong temporal association between SIP and the simultaneous treatment of ELBW infants with glucocorticoids and indomethacin.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Dexametasona/efeitos adversos , Modelos Animais de Doenças , Glucocorticoides/efeitos adversos , Indometacina/efeitos adversos , Perfuração Intestinal/induzido quimicamente , Animais , Animais Recém-Nascidos , Anti-Inflamatórios não Esteroides/administração & dosagem , Western Blotting , Dexametasona/administração & dosagem , Progressão da Doença , Sinergismo Farmacológico , Quimioterapia Combinada , Predisposição Genética para Doença , Glucocorticoides/administração & dosagem , Íleo/efeitos dos fármacos , Íleo/metabolismo , Indometacina/administração & dosagem , Perfuração Intestinal/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Peristaltismo/efeitos dos fármacos , Estenose Pilórica/etiologia , Fatores de RiscoRESUMO
Platelet and monocyte activation may contribute to hemolytic anemia, thrombocytopenia and renal failure associated with the hemolytic uremic syndrome (HUS) caused by Escherichia coli O157:H7. Since Shiga toxins (Stxs) and lipopolysaccharide (LPS) from this bacterium are implicated in the pathogenesis of HUS, we examined whether stimulation of THP-1 human monocytic cells by Shiga toxin 2 (Stx2) and LPS can lead to the activation of platelet function. We now show that Stx2 causedTHP-1 cells to release the chemokines IL-8, MDC, and RANTES and that the presence of LPS further stimulated this release. IL-8 was produced in greatest amount and was an effective co-agonist for inducing platelet aggregation. Primary human monocytes also released large amounts of IL-8 in response to LPS and Stx2. Factors released byTHP-1 cells exposed to Stx2 and LPS activated platelet function as evidenced by increased aggregation, serotonin secretion, P-selectin exposure and by the formation of stable platelet-monocyte aggregates. Our data therefore show that monocytes exposed to E.coli-derived Stx2 and LPS release factors which activate platelet function.
Assuntos
Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Agregação Plaquetária/fisiologia , Toxina Shiga II/farmacologia , Plaquetas/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Quimiocina CCL22 , Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Grânulos Citoplasmáticos/fisiologia , Humanos , Interleucina-8/metabolismo , Leucemia , Monócitos/citologiaRESUMO
BACKGROUND: It has been previously demonstrated that patients with reflux esophagitis exhibit a significant impairment in the secretion of salivary protective components versus controls. However, the secretion of salivary protective factors in patients with nonerosive reflux disease (NERD) is not explored. The authors therefore studied the secretion of salivary volume, pH, bicarbonate, nonbicarbonate glycoconjugate, protein, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α) and prostaglandin E2 in patients with NERD and compared with the corresponding values in controls (CTRL). METHODS: Salivary secretion was collected during basal condition, mastication and intraesophageal mechanical (tubing, balloon) and chemical (initial saline, acid, acid/pepsin, final saline) stimulations, respectively, mimicking the natural gastroesophageal reflux. RESULTS: Salivary volume, protein and TGF-α outputs in patients with NERD were significantly higher than CTRL during intraesophageal mechanical (P < 0.05) and chemical stimulations (P < 0.05). Salivary bicarbonate was significantly higher in NERD than CTRL group during intraesophageal stimulation with both acid/pepsin (P < 0.05) and saline (P < 0.01). Salivary glycoconjugate secretion was significantly higher in the NERD group than the CTRL group during chewing (P < 0.05), mechanical (P < 0.05) and chemical stimulation (P < 0.01). Salivary EGF secretion was higher in patients with NERD during mechanical stimulation (P < 0.05). CONCLUSIONS: Patients with NERD demonstrated a significantly stronger salivary secretory response in terms of volume, bicarbonate, glycoconjugate, protein, EGF and TGF-α than asymptomatic controls. This enhanced salivary esophagoprotection is potentially mediating resistance to the development of endoscopic mucosal changes by gastroesophageal reflux.
Assuntos
Refluxo Gastroesofágico , Saliva , Glândulas Salivares , Adulto , Dinoprostona/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Feminino , Refluxo Gastroesofágico/metabolismo , Refluxo Gastroesofágico/fisiopatologia , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Estimulação Física , Saliva/metabolismo , Eliminação Salivar , Glândulas Salivares/metabolismo , Glândulas Salivares/fisiopatologia , Cloreto de Sódio/metabolismo , Estimulação Química , Fator de Crescimento Transformador alfa/metabolismoRESUMO
PURPOSE: A significantly compromised epidermal growth factor (EGF) secretion by basal parotid saliva may contribute to the development of Barrett's esophagus (BE). The rate of secretion of EGF as well as a wide spectrum of protective factors in total basal and stimulated saliva in BE patients remains to be explored. We therefore studied the rate of secretion of salivary buffers, glycoconjugate, protein, EGF, transforming growth factor α (TGFα) and prostaglandin E2 (PGE2), evoked by esophago-salivary reflex, in patients with BE and controls (CTRL). MATERIAL/METHODS: Salivary secretion was collected during basal condition, mastication, and intraesophageal mechanical and chemical stimulations respectively, mimicking the natural gastroesophageal reflux scenario. RESULTS: Salivary pH in BE was significantly lower than in controls during mechanical (p<0.001) and chemical stimulations (p<0.001). Bicarbonate and protein outputs in BE were significantly lower during mechanical (p<0.05) and chemical stimulations (p<0.01). The non-bicarbonate and glycoconjugate outputs in BE were lower during chemical stimulation (p<0.05) and during mechanical (p<0.05) and chemical stimulations (p<0.05) respectively. The rate of salivary EGF output in BE was significantly lower during mechanical stimulation (p<0.05). We observed a higher TGFα output during mastication (p<0.05) and PGE2 secretion during basal and masticatory condition (p<0.05) in BE. CONCLUSIONS: Patients with BE demonstrated significantly compromised salivary pH and rate of secretion of bicarbonate, non-bicarbonate, glycoconjugate, protein and EGF. This impairment could potentially predispose to the development of accelerated esophageal mucosal injury. Potential restoration of this impairment by masticatory stimulation of salivary secretion using sugarless chewing gum justifies further clinical exploration.
Assuntos
Esôfago de Barrett/fisiopatologia , Regulação para Baixo , Esôfago/fisiopatologia , Reflexo Anormal , Glândulas Salivares/fisiopatologia , Salivação , Adulto , Idoso , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Saliva/química , Saliva/metabolismo , Glândulas Salivares/metabolismoRESUMO
BACKGROUND: Mucosal apoptosis is the initiating event in models of necrotizing enterocolitis (NEC) within rodents. It is possible there are species-specific differences that make apoptosis a more prominent feature of NEC in rodents than in humans. HYPOTHESIS: A lower threshold for mucosal apoptosis in the rodent distal intestine might have evolutionary advantages (via enhanced opsonization with the neonatal Fc receptor [FcRn]), since many short-gestation mammals are comparatively premature (histomorphologically) but are protected from NEC by breast milk. METHODS: We utilized a rat intestinal epithelial cell (IEC-18) model to determine if cell death alters FcRn - IgG binding, and rodent models of NEC to determine if cell death results in increased opsonization of IgG. Cultured IEC-18 cells were treated with H2O2 and analyzed. Neonatal Sprague-Dawley rats were cold and hypoxia stressed and intestinal sections were frozen for analysis. RESULTS: IgG binding was increased in H2O2-treated cells. Co-incubation of treated cells with either insulin-like growth factor or tunicamycin decreased IgG binding. Sprague-Dawley rats formula fed with exogenous bacteria showed a significant decrease in intestinal FcRn mRNA but increased ileal IgG binding. CONCLUSIONS: We speculate that FcRn plays a role in passive opsonization and subsequent bacterial pathogen clearance, making rodents resistant to NEC.
RESUMO
Obesity and overweight are linked to a wide range of medical conditions, such as hypertension, diabetes mellitus, osteoarthritis, obstructive sleep apnea, and coronary artery disease. Overweight and obese patients who are unable to lose weight with diet alone can benefit from well-structured exercise. Potentially, an individual exercise prescription can become one of the most important components of an obesity treatment program, along with an appropriate diet. Short-term (<6 months of duration) interventions consisting of exercise combined with appropriate diet and counseling can produce a significant weight loss. No consensus exists on the amount of physical activity necessary to maintain the weight loss achieved during a short-term intervention. Long-term intervention is frequently influenced by weight regain related to complex interactions between physiologic and psychosocial factors.
Assuntos
Exercício Físico/fisiologia , Obesidade/terapia , Adulto , Idoso , Índice de Massa Corporal , Pré-Escolar , Humanos , Obesidade/fisiopatologia , Redução de PesoRESUMO
Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.
Assuntos
Quimiocinas/metabolismo , Endotélio Vascular/imunologia , Lipopolissacarídeos/farmacologia , Agregação Plaquetária , Toxina Shiga II/farmacologia , Plaquetas/metabolismo , Capilares/citologia , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Escherichia coli O157/patogenicidade , Humanos , Selectina-P/farmacologia , Serotonina/metabolismoRESUMO
The in vitro oxidation of low-density lipoprotein (LDL) by hypochlorous acid produces a modified form (HOCl-LDL) capable of stimulating platelet function. We now report that HOCl-LDL is highly effective at inducing platelet function, causing stable aggregation and alpha-granule secretion. Such stimulation depended on the presence of low levels of primary agonists such as adenosine diphosphate (ADP) and thrombin, or others like epinephrine (EPI) and macrophage-derived chemokine (MDC, CCL22). Agonist levels, which by themselves induced little or reversible aggregation, caused strong stable aggregation when combined with low levels of HOCl-LDL. Platelet activation by HOCl-LDL and ADP (1 microM) caused P-selectin (CD62P) exposure, without serotonin or adenosine triphosphate (ATP) secretion. Intracellular calcium levels rose slowly (from 100 to 200 nM) in response to HOCl-LDL alone and rapidly when combined with ADP to about 300 nM. p38 mitogen-activated protein kinase (MAPK) became phosphorylated in response to HOCl-LDL alone. This phosphorylation was not blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, which reduced the extent of aggregation and calcium increase. However, the p38 MAPK inhibitor SB203580 blocked platelet aggregation and phosphorylation of p38 MAPK. These findings suggest that HOCl-LDL exposed during atherosclerotic plaque rupture, coupled with low levels of primary agonists, can rapidly induce extensive and stable thrombus formation.