RESUMEN
Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was â¼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.
Asunto(s)
Fosfopiruvato Hidratasa , Tartronatos , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/metabolismo , Hidroliasas/química , Racemasas y Epimerasas/metabolismo , CinéticaRESUMEN
Sepsis is a global disease burden, and approximately 40% of cases develop acute lung injury (ALI). Bone marrow mesenchymal stromal cells (BMSCs) and their exosomes are widely used in treating a variety of diseases including sepsis. As an acute phase protein, serum amyloid A1 (SAA1) regulates inflammation and immunity. However, the role of SAA1 in BMSCs-exosomes in septic lung injury remains to be elucidated. Exosomes derived from serum and BMSCs were isolated by ultracentrifugation. SAA1 was silenced or overexpressed in mouse BMSCs using lentiviral plasmids, containing either SAA1-targeting short interfering RNAs or SAA1 cDNA. Sepsis was induced by cecal ligation and puncture (CLP). LPS was used to induce ALI in mice. Mouse alveolar macrophages were isolated by flow cytometry. Levels of SAA1, endotoxin, TNF-α, and IL-6 were measured using commercial kits. LPS internalization was monitored by immunostaining. RT-qPCR or immunoblots were performed to test gene and protein expressions. Serum exosomes of patients with sepsis-induced lung injury had significantly higher levels of SAA1, endotoxin, TNF-α, and IL-6. Overexpression of SAA1 in BMSCs inhibited CLP- or LPS-induced lung injury and decreased CLP- or LPS-induced endotoxin, TNF-α, and IL-6 levels. Administration of the SAA1 blocking peptide was found to partially inhibit SAA1-induced LPS internalization by mouse alveolar macrophages and reverse the protective effect of SAA1. In conclusion, BMSCs inhibit sepsis-induced lung injury through exosomal SAA1. These results highlight the importance of BMSCs, exosomes, and SAA1, which may provide novel directions for the treatment of septic lung injury.
Asunto(s)
Lesión Pulmonar Aguda , Células Madre Mesenquimatosas , Sepsis , Proteína Amiloide A Sérica , Animales , Ratones , Lesión Pulmonar Aguda/inducido químicamente , Células de la Médula Ósea/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteína Amiloide A Sérica/genética , ExosomasRESUMEN
Aims: Dexmedetomidine (Dex) as a highly selective α2-adrenoceptor agonist, was widely used anesthetic in perioperative settings, whether Dex induces cardiac hypertrophy during perioperative administration is unknown. Methods: The effects of Dex on cardiac hypertrophy were explored using the transverse aortic constriction model and neonatal rat cardiomyocytes. Results: We reported that Dex induces cardiomyocyte hypertrophy with activated ERK, AKT, PKC and inactivated AMPK in both wild-type mice and primary cultured rat cardiomyocytes. Additionally, pre-administration of Dex protects against transverse aortic constriction induced-heart failure in mice. We found that Dex up-regulates the activation of ERK, AKT, and PKC via suppression of AMPK activation in rat cardiomyocytes. However, suppression of mitochondrial coupling efficiency and membrane potential by FCCP blocks Dex induced AMPK inactivation as well as ERK, AKT, and PKC activation. All of these effects are blocked by the α2-adrenoceptor antagonist atipamezole. Conclusion: The present study demonstrates Dex preconditioning induces cardiac hypertrophy that protects against heart failure through mitochondria-AMPK pathway in perioperative settings.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Cardiomegalia/inducido químicamente , Dexmedetomidina/farmacología , Insuficiencia Cardíaca/prevención & control , Agonistas de Receptores Adrenérgicos alfa 2/uso terapéutico , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/administración & dosificación , Células Cultivadas , Dexmedetomidina/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Cultivo Primario de Células , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Sepsis is a significant contributor to both intensive care unit (ICU) admissions and mortality among patients in ICU, with a rising prevalence of obesity. There is a lack of extensive research on the correlation between TyGI and findings in patients with sepsis, especially in obese patients. METHODS: This study used a retrospective cohort design and included patients with sepsis (≥18 years) from the Medical Information Mart for Intensive Care IV database. The association between TyGI and outcome was examined using multivariable logistic regression analysis. RESULTS: 8,840 patients with sepsis were included in the analysis. The in-ICU mortality rate was 9.7%. Non-survivors exhibited significantly greater TyGI levels than survivors [9.19(8.76-9.71) vs. 9.10(8.67-9.54), p < 0.001]. The adjusted multivariate regression model showed that elevated TyGI values were linked to a greater likelihood of death in ICU (odds ratio [OR] range 1.072-1.793, p < 0.001) and hospital (OR range 1.068-1.445, p = 0.005). Restricted Cubic Spline analysis revealed a nonlinear association between TyGI and in-ICU and in-hospital mortality risks within specified ranges. Subgroup analysis revealed interaction effects in the general obesity, abdominal obesity, and impaired fasting glucose subgroups (p = 0.014, 0.016, and < 0.001, respectively). CONCLUSION: TyGI was associated with an increased sepsis-related short-term mortality risk and adverse outcomes after ICU admission.
Asunto(s)
Glucemia , Mortalidad Hospitalaria , Unidades de Cuidados Intensivos , Obesidad , Sepsis , Triglicéridos , Humanos , Sepsis/mortalidad , Sepsis/metabolismo , Estudios Retrospectivos , Masculino , Femenino , Persona de Mediana Edad , Obesidad/mortalidad , Obesidad/metabolismo , Obesidad/complicaciones , Anciano , Triglicéridos/sangre , Triglicéridos/metabolismo , Glucemia/análisis , Glucemia/metabolismo , AdultoRESUMEN
BACKGROUND: Endotracheal intubation (ETI) is a life-saving procedure taught to medical students. We examined the influence of the order of teaching ETI through direct laryngoscopy (DL) and video laryngoscopy (VL) on learning by measuring the intubation time and learning curve of trainees, in order to explore ways to improve ETI performance. METHODS: Twenty trainees were randomly divided into 2 groups. In the DL-first group, trainees used DL to perform ETI 10 times and then used VL 10 times, while the order was reversed in the VL-first group. Intubation time, number of intubation attempts, the Cormack-Lehane (CL) classification, and adverse events were recorded. The primary outcome was the cumulative summation (CUSUM). The CUSUM equation is defined as (Equation is included in full-text article.), where ct is the cumulative sum. RESULTS: ETI was attempted on 400 patients. The difference in the mean times for the first 10 intubations between the 2 groups was not significant (Pâ>â.05). Mean intubation time for second series in the DL-first group was significantly shorter than that of the first series (Pâ<â.05), while there were no differences between the 2 series in the VL-first group (Pâ>â.05). The mean intubation time in the second series of the DL-first group was shorter than for the first series of the VL-first group (Pâ<â.05), while the mean intubation time of the first series by the DL-first group did not differ from the second series by the VL-first group (Pâ>â.05). Eighteen attempts were required to achieve an 80% intubation success rate for the DL-first group, while more than 20 attempts were required for the trainees in the VL-first group. CONCLUSION: We consider that teaching trainees DL for tracheal intubation first. CLINICAL TRIAL NUMBER: ChiCTR-OOR-16008364.