Asunto(s)

Esclerosis Amiotrófica Lateral , Mutación , Proteína FUS de Unión a ARN , Humanos , Femenino , Proteína FUS de Unión a ARN/genética , Esclerosis Amiotrófica Lateral/genética , Lactante , Exones , Resultado Fatal , Heterocigoto , Análisis Mutacional de ADN

Asunto(s)

Atrofia Muscular Espinal , Humanos , Masculino , Femenino , Lactante , Preescolar , Atrofia Muscular Espinal/terapia , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofias Musculares Espinales de la Infancia/terapia , Atrofias Musculares Espinales de la Infancia/genética , Atrofias Musculares Espinales de la Infancia/diagnóstico , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Niño , Exones , Oligonucleótidos

RESUMEN

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.

Asunto(s)

Anomalías Múltiples , Enfermedades del Desarrollo Óseo , Discapacidad Intelectual , Atrofia Muscular Espinal , Distrofia Muscular de Duchenne , Anomalías Dentarias , Humanos , Estudios Retrospectivos , Discapacidad Intelectual/genética , Enfermedades del Desarrollo Óseo/complicaciones , Anomalías Dentarias/complicaciones , Facies , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/complicaciones , Atrofia Muscular Espinal/complicaciones , Proteínas Portadoras , Proteínas Nucleares

RESUMEN

Objective: To analyze the follow-up and clinical effect of multidisciplinary treatment on the children with spinal muscular atrophy (SMA). Methods: The clinical data including nutritional status, respiratory function, bone health and motor function of 45 children with SMA who received multidisciplinary management 1-year follow-up in the Children's Hospital, Zhejiang University School of Medicine from July 2019 to October 2021 were retrospectively collected. Comparisons before and after management were performed using paired-samples t-test or Wilcoxon rank-sum test, etc. Results: The age of 45 patients (25 boys and 20 girls) was 50.4 (33.6, 84.0) months at the enrollment, with 6 cases of type 1, 22 cases of type 2, and 17 cases of type 3 respectively. After the multidisciplinary management, the cases of SMA patients with malnutrition decreased from 22 to 12 (P=0.030), the level of vitamin D were significantly increased ((45±17) vs. (48±14) nmol/L, t=-4.13, P<0.001). There was no significant difference in the forced vital capacity %pred, the forced expiratory volume at 1 second %pred, and the peak expiratory flow %pred ((76±19)% and (76±21)%, (81±18)% and (79±18)%, (81±21)% and (78±17)%; t=-0.24, 1.36, 1.21; all P>0.05). The Cobbs angle of scoliosis also improved significantly (8.0°(0°, 13.0°) vs. 10.0°(0°, 18.5°), Z=-3.01, P=0.003). The Hammersmith functional motor scale expanded scores of children with SMA type 2 and type 3 both showed significant elevation (11.0 (8.0, 18.0) vs. 11.0 (5.0, 18.5) scores, 44.0 (36.5, 53.0) vs. 44.0 (34.0, 51.5) scores, Z=2.44, 3.11, P=0.015, 0.002). Conclusion: Multidisciplinary management is beneficial for delaying the progression of the multi-system impairments of SMA patients, such as malnutrition, restrictive ventilation dysfunction and scoliosis.

Asunto(s)

Desnutrición , Atrofia Muscular Espinal , Escoliosis , Niño , Masculino , Femenino , Humanos , Preescolar , Estudios Retrospectivos , Estudios de Seguimiento

RESUMEN

Objective: To explore the clinical efficacy of disease-modifying drug nusinersen on children with spinal muscular atrophy. Methods: The baseline and longitudinal clinical data of 15 children who were treated with nusinersen in the Children's Hospital, Zhejiang University School of Medicine from October 2019 to October 2021 were retrospectively collected. The general data (gender, age, genotype, and clinical classification, etc.), motor function, nutritional status, scoliosis and respiratory function were analyzed. Wilcoxon rank-sum test was used for comparing multi-system conditions before and after treatment. Results: The age of 15 cases (7 males, 8 females) was 6.8 (2.8, 8.3) years, with 2 cases of type 1, 6 cases of type 2, and 7 cases of type 3 respectively, and the course of disease was 55.0 (21.0, 69.0) months. After 9.0 (9.0, 24.0) months of treatment, the motor function scale evaluations of the Hammersmith neurological examination section 2 (13.0 (7.0, 23.0) vs. 18.0 (10.0, 25.0) scores, Z=-2.67, P=0.018) of 15 children, the Hammersmith functional motor scale expanded (38.0 (18.5, 45.5) vs. 42.0 (23.0, 51.0) scores, Z=-2.38, P=0.018), and the revised upper limb module (27.0 (19.5, 32.0) vs. 33.0 (22.5, 35.5) scores, Z=-2.52, P=0.012) of children with type 2 and 3 had significantly improved. Thirteen patients achieved clinically significant motor function improvement, and 2 of them had kept stable scale scores. Subjective reports also indicated that the muscle strength and daily exercise ability of these children improved after treatment, and no serious adverse reactions were reported. Supplemented by the multi-disciplinary team management, the levels of some indicators such as Cobbs angle of scoliosis and forced vital capacity all had significantly improved (all P<0.05). Conclusions: Nusinersen can improve the motor function of patients with 5q spinal muscular atrophy, which is also proved safe to be used in children. The drug treatment supplemented by the multi-disciplinary team management is helpful to improve the multi-system function of the children with spinal muscular atrophy.

Asunto(s)

Atrofia Muscular Espinal , Oligonucleótidos , Escoliosis , Atrofias Musculares Espinales de la Infancia , Niño , Preescolar , China , Femenino , Estudios de Seguimiento , Humanos , Masculino , Atrofia Muscular Espinal/tratamiento farmacológico , Oligonucleótidos/uso terapéutico , Estudios Retrospectivos , Escoliosis/tratamiento farmacológico , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico

Asunto(s)

Atrofia Muscular Espinal , Humanos , Atrofia Muscular Espinal/tratamiento farmacológico

RESUMEN

Fish scales serve as a dermal armor that provides protection from physical injury. Owing to a number of outstanding properties, fish scales are inspiring new concepts for layered engineered materials and next-generation flexible armors. Although past efforts have primarily focused on the structure and mechanical behavior of ontogenetic scales, the structure-property relationships of regenerated scales have received limited attention. In the present study, common carp (Cyprinus carpio) acquired from the wild were held live in an aquatic laboratory at 10°C and 20°C. Ontogenetic scales were extracted from the fish for analysis, as well as regenerated scales after approximately 1 year of development and growth. Their microstructure was characterized using microscopy and Raman spectroscopy, and the mechanical properties were evaluated in uniaxial tension to failure under hydrated conditions. The strength, strain to fracture and toughness of the regenerated scales were significantly lower than those of ontogenetic scales from the same fish, regardless of the water temperature. Scales that regenerated at 20°C exhibited significantly higher strength, strain to fracture and toughness than those regenerated at 10°C. The regenerated scales exhibited a highly mineralized outer layer, but no distinct limiting layer or external elasmodine; they also possessed a significantly lower number of plies in the basal layer than the ontogenetic scales. The results suggest that a mineralized layer develops preferentially during scale regeneration with the topology needed for protection, prior to the development of other qualities.

Asunto(s)

Carpas , Animales , Espectrometría Raman , Temperatura , Agua

RESUMEN

An advanced in vitro cervical tumor model was established by 3D printing to study the epithelial-to-mesenchymal transition (EMT), which is a very important stage of dissemination of carcinoma leading to metastatic tumors. A HeLa/hydrogel grid construct composed of gelatin, alginate, Matrigel and HeLa cells was fabricated by forced extrusion in a layer-by-layer fashion. HeLa cells rapidly proliferated, formed spheroids and presented tumorigenic characteristic in the 3D-printed structure. With the supplement of TGF-ß, aggregated HeLa cells started to disintegrate, and some of them changed into fibroblast-like spindle morphology, which indicated that EMT was induced. The down-regulation of epithelial marker E-cadherin, and up-regulation of mesenchymal markers such as snail, vimentin and N-cadherin were all observed in the 3D-printed model, and performed differently in 3D and 2D models. The TGF-ß induced EMT was inhibited by the treatment of disulfiram and EMT pathway inhibitor C19 in a dose dependent manner, showing great potential for future studies of a therapeutic program towards cervical tumor metastasis.

Asunto(s)

Transición Epitelial-Mesenquimal/efectos de los fármacos , Impresión Tridimensional , Factor de Crecimiento Transformador beta/farmacología , Neoplasias del Cuello Uterino/patología , Biomarcadores de Tumor/metabolismo , Forma de la Célula/efectos de los fármacos , Disulfiram/farmacología , Femenino , Células HeLa , Humanos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología

RESUMEN

Osteoporosis and atherosclerosis are two prevalent major healthcare concerns that frequently coexist. The clinical outcome of 5590 consecutive subjects who underwent coronary artery calcium (CAC) scanning and thoracic bone mineral density (BMD) measurement was assessed. A significant link between low BMD levels and CAC with increased risk of mortality in both genders across ethnicities noted. INTRODUCTION: While a relation of CAC with lower levels of BMD reported previously; it is unclear whether low levels of BMD would be an independent risk factor for CAC and mortality. This study investigated the relation of BMD levels with CAC and mortality in both genders across ethnicities. METHODS: This study consisted of 5590 consecutive at-risk subjects without known coronary artery disease (CAD), age 57 ± 12, and 69% male, who underwent non-enhanced cardiac computed tomography, and were followed for mean of 8 years. The subjects' CAC (Agatston score) and thoracic BMD levels (mg/cm3) were measured. CAC stratified based on the severity to CAC 0, 1-100, 101-400, and 400+. Low-BMD levels defined as BMD levels below median (180 mg/cm3). Physician verified that all-cause mortality was assessment hard-endpoint. Multivariate regression analysis, adjusted for age, gender, and other cardiovascular risk factors, was used to assess the relationship between BMD and CAC. RESULTS: The BMD levels were proportionally lowering with the severity of CAC in both genders, especially in postmenopausal women (p < 0.05). The risk of each standard deviation reduce in BMD levels increased with the severity of CAC, as compared to CAC = 0 across ethnicities (p < 0.05). Low BMD levels were an independent predictor of mortality and event-free survival rate decreased from 99% in those within normal BMD levels to 93% in those with low BMD levels (p = 0.0001). Furthermore, a significant link between low BMD levels and CAC > 0 with increased risk of mortality was noted (p = 0.0001). The relative risk of death was 2.8, 5.9, and 14.3-folds higher in CAC 1-100, 101-400, and 400+ with low BMD levels, compared to CAC = 0 and within normal BMD levels, respectively (p < 0.05). CONCLUSIONS: The lower BMD levels are independently associated with the severity of CAC that predicts mortality.

Asunto(s)

Densidad Ósea/fisiología , Enfermedad de la Arteria Coronaria/mortalidad , Osteoporosis/mortalidad , Calcificación Vascular/mortalidad , Adulto , Anciano , Enfermedad de la Arteria Coronaria/complicaciones , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/complicaciones , Osteoporosis/fisiopatología , Factores de Riesgo , Índice de Severidad de la Enfermedad , Vértebras Torácicas/diagnóstico por imagen , Vértebras Torácicas/fisiopatología , Tomografía Computarizada por Rayos X , Estados Unidos/epidemiología , Calcificación Vascular/complicaciones , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/fisiopatología

RESUMEN

Many epidemiological reports stated a strong association between maternal infection and development of cerebral palsy, which is a major cause of cognitive impairment. The pathophysiological mechanism of intrauterine inflammation is complex. Recently, it was demonstrated that inflammation has a modulating effect on adult neurogenesis. In this study, we discovered the effect of maternal infection to hippocampal neuronal apoptosis, proliferation and differentiation, and cognitive development in the developing brains of neonatal rats. Morris water maze test was used to assess learning and memory. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine neuronal apoptosis, immunostaining was conducted to assess neurogenesis, and Western blot for extracellular signal-regulated kinase (ERK), cyclic AMP responsive element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Results demonstrated that maternal infection increased neuronal apoptosis and significantly impaired spatial learning and memory ability. Maternal infection significantly increased cell proliferation, accompanied by an increased expression of ERK (P3-P7), CREB (P3-P7) and BDNF (P3). On P28, there was no significant difference of cell survival and differentiation in two groups. These results suggest that variation in ERK activity and subsequent expression of its downstream targets, including CREB and BDNF might contribute, at least partially, to modulation of inflammation related cell proliferation, survival and differentiation. Maternal infection increased hippocampal neuronal apoptosis and affected cell proliferation and differentiation in neonatal rats, which may be regarded as an etiological factor in cognitive development impairment.

RESUMEN

Femtosecond and nanosecond lasers were compared for ablating brass alloys. All operating parameters from both lasers were equal except for the pulse duration. The ablated aerosol vapor was collected on silicon substrates for particle size measurements or sent into an inductively coupled plasma mass spectrometer. The diameters and size distribution of particulates were measured from scanning electron microscope (SEM) images of the collected ablated aerosol. SEM measurements showed that particles ablated using nanosecond pulses were single spherical entities ranging in diameter from several micrometers to several hundred nanometers. Primary particles ablated using femtosecond ablation were approximately 100 nm in diameter but formed large agglomerates. ICPMS showed enhanced signal intensity and stability using femtosecond compared to nanosecond laser ablation.

RESUMEN

Electron number density and temperature were determined from laser-induced plasmas produced by irradiating Al-Cu-Fe targets of a quasicrystal and of an alloy of similar composition. The Al(I) atomic emission spectra of the two systems were measured as a function of the distance from the target and of the time delay after laser irradiation. Differences of plasma characteristics were observed for laser ablation of quasicrystal and alloy targets, and the results were interpreted on the basis of different plasma formation mechanisms for the two systems.

Asunto(s)

Aleaciones/química , Aluminio/química , Cobre/química , Hierro/química , Análisis Espectral/métodos , Cristalización , Cristalografía , Rayos Láser

RESUMEN

RATIONALE AND OBJECTIVES: Our purpose was to investigate the motion characteristics of the coronary arteries and determine optimal electrocardiographic (ECG) trigger time during the cardiac cycle to minimize motion artifacts. METHODS: Contrast-enhanced multislice movie studies of electron beam tomography (EBT) images were performed on 70 subjects. The EBT datasets, which covered an entire cardiac cycle at 58-ms intervals, were acquired for a short-axis view of the heart with ECG triggering. The pixel values along x and y axes were measured at multiple intervals during the cardiac cycle to establish the motion distance and velocity of three major coronary arteries. RESULTS: Coronary artery motion varied greatly throughout the cardiac cycle in three major coronary arteries and increased with the patient's baseline heart rate. The greatest and lowest velocities of coronary arterial movement during the cardiac cycle were determined. Based on the lowest velocity of right coronary artery movement during the cardiac cycle, the optimal ECG trigger times were located at approximately 35% (31.4%-37.6%) or 70% (68.7%-71.4%) of the R-R interval in patients whose resting heart rate was < or =70 beats per minute (bpm); at 50% (47.2%-61.1%) of the R-R interval in the 71- to 100-bpm group; and at 55% (52.8%-59.1%) of the R-R interval in the >100-bpm group. Our data demonstrated that the motion characteristics of the left circumflex artery were quite similar to those of the right coronary artery and that the left anterior descending coronary artery had no significant differences in motion throughout the cardiac cycle. A minimum scan speed of 35.4 to 75.5 ms per slice is needed to completely diminish cardiac motion artifacts (in-plane coronary artery motion with <1-mm displacement). CONCLUSIONS: For coronary artery screening, the optimal ECG trigger time should be determined according to the patient's heart rate, thus greatly reducing motion and motion artifacts during 100-ms acquisitions.

Asunto(s)

Vasos Coronarios/fisiología , Electrocardiografía , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Corazón/fisiología , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.

Asunto(s)

Carboxipeptidasas/sangre , Carboxipeptidasas/metabolismo , Precursores Enzimáticos/sangre , Precursores Enzimáticos/metabolismo , Fibrinolisina/metabolismo , Fibrinólisis , Animales , Arginina/metabolismo , Sitios de Unión , Western Blotting , Carboxipeptidasa B , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Fibrinolisina/farmacología , Fibrinólisis/efectos de los fármacos , Fibrinolíticos/farmacología , Glicosaminoglicanos/metabolismo , Hemostáticos/farmacología , Heparina/metabolismo , Heparina/farmacología , Humanos , Inmunoglobulina G/metabolismo , Cinética , Conejos , Trombina/farmacología , Trombomodulina/metabolismo , Factores de Tiempo , Activador de Tejido Plasminógeno/metabolismo

RESUMEN

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.

Asunto(s)

Anticoagulantes/farmacología , Inhibidores del Factor Xa , Hormonas de Invertebrados/farmacología , Fragmentos de Péptidos/farmacología , Tromboplastina/metabolismo , Secuencia de Aminoácidos , Animales , Anticoagulantes/química , Células COS , Catálisis , Bovinos , Humanos , Hormonas de Invertebrados/química , Cinética , Datos de Secuencia Molecular , Tiempo de Tromboplastina Parcial , Fragmentos de Péptidos/química , Protrombina/metabolismo , Spodoptera

RESUMEN

A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.

Asunto(s)

Péptidos/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Clonación Molecular , ADN Recombinante , Vectores Genéticos , Péptidos y Proteínas de Señalización Intercelular , Factor de Apareamiento , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Garrapatas/genética

RESUMEN

The interaction of thrombin with several potent and selective alpha-ketoamide transition state analogs was characterized. L-370, 518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (Ki = 90 pM) and selective (>10(4)-fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed that k-1, the rate constant for dissociation of EI1, was comparable to k2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s-1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nM) largely due to an increase in k-1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1' N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K-1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pM) than L-370,518 due to its 16-fold higher k2 and 10-fold lower k-2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K-1 value for its alpha-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the Ki value for L-372,011 and the K-1 value for its alpha-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the alpha-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the alpha-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.

Asunto(s)

Inhibidores de Serina Proteinasa/farmacología , Trombina/antagonistas & inhibidores , Sitios de Unión , Unión Competitiva , Catálisis , Análisis de Inyección de Flujo , Colorantes Fluorescentes , Cinética , Modelos Químicos , Oligopéptidos/farmacología , Inhibidores de Serina Proteinasa/química

RESUMEN

A low half-wave voltage V(pi) of 1.57 V was obtained with a 2-cm-long birefringent polymer waveguide modulator at a wavelength of 1.3 microm by use of a modulator design with a constant-bias electric field and a high-microbeta chromophore. The design allows the maximum achievable electro-optic coefficient of the material to be utilized. This electro-optic coefficient can be more than twice as high as the residue value that is used by conventional modulator designs, after fast partial relaxation following poling.

RESUMEN

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Asunto(s)

Antitrombinas/síntesis química , Antitrombinas/farmacología , Dipéptidos/síntesis química , Dipéptidos/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Trombina/antagonistas & inhibidores , Administración Oral , Animales , Antitrombinas/farmacocinética , Disponibilidad Biológica , Cristalografía por Rayos X , Dipéptidos/farmacocinética , Perros , Cinética , Piridinas/farmacocinética , Ratas , Relación Estructura-Actividad , Trombina/metabolismo

RESUMEN

OBJECTIVES: Electrocardiographic (ECG) trigger records obtained during cardiac ultrafast computed tomography (UFCT) scanning were analyzed to estimate the variability in heart rate and ECG trigger interval to develop a protocol that would allow the development of better ECG triggering software. METHODS: One-hundred-eighteen patients underwent cardiac UFCT imaging for diagnostic purposes. All subjects were divided into three groups according to the heart rate and ECG trigger condition. Thirty slices were obtained in the high-resolution volume mode for each patient. RESULTS: A decrease in heart rate and ECG trigger interval was found during image acquisition of the first four slices in all three groups. The nadir of the heart rate occurred during acquisition of the 4th slice, 5.3, 3.5, and 5.6 beats per minute less than the initial heart rate in groups 1, 2, and 3 respectively, with a 6.9%, 2.8%, and 5.0% shorter ECG trigger interval (p < .001, p = .08, p < .05, respectively). From the 4th to the 30th slices, heart rate and ECG trigger interval progressively increased, but less variability was found in the last 20 slices in all three groups. CONCLUSIONS: Significant variation in heart rate and ECG trigger interval was seen during 30-level cardiac UFCT imaging, especially during image acquisition of the first four slices (approximately 1-6 seconds after breatholding). This can result in scanning during the suboptimal phase of the cardiac cycle by the current UFCT triggering software. A delay in the initiation of scanning to approximately 6 to 10 seconds after breatholding would result in imaging during a time when the heart rate is relatively stable, and a smaller variability in ECG trigger interval occurs. Recalculation of the required delay before each heart beat may improve the precision of ECG triggering.

Asunto(s)

Electrocardiografía/métodos , Frecuencia Cardíaca/fisiología , Corazón/diagnóstico por imagen , Interpretación de Imagen Asistida por Computador/métodos , Tomografía Computarizada por Rayos X/métodos , Femenino , Corazón/fisiología , Humanos , Masculino , Persona de Mediana Edad