RESUMEN
Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Inhibidores Enzimáticos/química , Femenino , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ubiquitina/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Understanding the catalysis of oxidative reactions by TAML activators of peroxides, i. e. iron(III) complexes of tetraamide macrocyclic ligands, advocated a spectrophotometric procedure for quantifying the catalytic activity of TAMLs for colorless targets (kII ', M-1 s-1 ), which is incomparably more advantageous in terms of time, cost, energy, and ecology than NMR, HPLC, UPLC, GC-MS and other similar techniques. Dyes Orange II or Safranin O (S) are catalytically bleached by non-excessive amount of H2 O2 in the presence of colorless substrates (S1 ) according to the rate law: -d[S]/dt=kI kII [H2 O2 ][S][TAML]/(kI [H2 O2 ]+kII [S]+kII '[S1 ]). The bleaching rate is thus a descending hyperbolic function of S1 : v=ab/(b+[S1 ]). Values of kII ' found from a and b for phenol and propranolol with commonly used TAML [FeIII {o,o'-C6 H4 (NCONMe2 CO)2 CMe2 }2 (OH2 )]+ are consistent with those for S1 (phenol, propranolol) obtained directly by UPLC. The study sends vital messages to enzymologists and environmentalists.
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BACKGROUND: Lymphedema is a common complication of breast cancer treatment that affects one in five breast cancer survivors, yet there is no reliable method to detect lymphedema in the subclinical range. The objective of this study was to determine the feasibility and reliability of using an infrared 3D scanning device (ISD) as a peri-operative limb volume measurement tool. METHODS: Fifteen patients were analyzed based on inclusion criteria. Peri-operative measurements were obtained using tape measure and an ISD. Volumes were calculated using a standard algorithm for tape measure and a custom algorithm for ISD measurements. Linear regression models were used to assess ISD and tape measurement volume and circumference correlation. One-way ANOVA was used to compare change in percent difference at set time points post-operatively (2-3 weeks, 4-6 weeks, and 7-12 weeks) for both ISD and tape measure. t tests for unequal variances with the Bonferroni correction were performed among these groups. RESULTS: There is a positive linear correlation (R2 = 0.8518) between absolute volume measurements by the ISD and tape measure. Analyses over 2-10 weeks post-operatively showed that the ISD was able to detect volume changes in both the unaffected and the affected arm. Furthermore, the affected arm tended to have a greater increase in volume in the majority of patients, indicating these patients could be at risk for lymphedema. CONCLUSIONS: Technology utilizing infrared 3D scanners can reliably measure limb volume pre- and post-treatment similarly to tape measure in a small sample of patients. Further research using 3D scanning technology with a longer follow up is warranted.
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Neoplasias de la Mama , Linfedema , Brazo , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/cirugía , Humanos , Pronóstico , Reproducibilidad de los ResultadosRESUMEN
Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD) or 9-hydroperoxy-10E,12Z-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl. Acad. Sci. USA96 (1999) 4192-4197) have identified an important residue, corresponding to phe-557 in soybean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of 13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced 33% 9-HPOD (S:Râ¯=â¯87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12% with the F557V,R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to 13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence that 13(S)-HPOD arises from tail-first binding.
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Ácidos Grasos Insaturados/metabolismo , Glycine max/enzimología , Lipooxigenasa/metabolismo , Sitios de Unión , Catálisis , Deuterio/química , Ácidos Grasos Insaturados/química , Ácidos Linoleicos/química , Peróxidos Lipídicos/química , Lipooxigenasa/genética , Mutación , Oxidación-Reducción , Fosfatidilcolinas/química , Unión Proteica , EstereoisomerismoRESUMEN
Phenotypic plasticity allows a plant cell to alter its structure and function in response to external pressure. This adaptive phenomenon has also been important in the evolution of plants including the emergence of land plants from a streptophyte alga. Penium margaritaceum is a unicellular zygnematophyte (i.e., the group of streptophyte algae that is sister to land plants) that was employed in order to study phenotypic plasticity with a focus on the role of subcellular expansion centers and the cell wall in this process. Live cell fluorescence labeling, immunofluorescence labeling, transmission electron microscopy, and scanning electron microscopy showed significant subcellular changes and alterations to the cell wall. When treated with the actin-perturbing agent, cytochalasin E, cytokinesis is arrested and cells are transformed into pseudo-filaments made of up to eight or more cellular units. When treated with the cyclin-dependent kinase (CDK) inhibitor, roscovitine, cells converted to a unique phenotype with a narrow isthmus zone.
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Fenotipo , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Pared Celular/metabolismo , Roscovitina/farmacología , Citocinesis/efectos de los fármacos , Actinas/metabolismoRESUMEN
Background: There are many techniques of monitoring leg lymphedema during physical therapy. Taking volumetric measurements with a tape measure is among the most common clinically, and changes in volume are typically used to measure therapy efficacy. This study shows how the Kinect infrared (IR) sensor with custom algorithms can assess leg circumferences and volumes comparable with tape measurements taken by a trained therapist while exploring regional leg changes to determine uniformity of change. Methods and Results: Leg volumes were measured in 38 lymphedema patients using the tape measure circumference method and the Kinect IR system. Changes in circumferences in various leg regions over the course of therapy were analyzed in 23 patients. The leg circumferences (R2 = 0.9522) and volumes ( R2 = 0.9847) strongly correlated between the two methods. The Bland-Altman analysis indicated a circumference percent different bias of 1.6 (6.2%), requiring a minor correction factor between the two methods. Over the course of therapy, patients with a reduction in leg volume, defined as a change >6.5% have greater reduction most distal to the body. Conclusion: The Kinect IR system explored can be used clinically for leg volume measurements to monitor leg lymphedema patients over the course of their therapy. Implementing analysis of regional leg changes can better inform physical therapy to improve efficacy of treatment.
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Pierna , Linfedema , Modalidades de Fisioterapia , Algoritmos , Humanos , Rayos Infrarrojos , Extremidad Inferior , Linfedema/diagnósticoRESUMEN
Background: There are various methods of detecting and monitoring arm lymphedema after breast cancer (BC) treatment. Using volumetric measurements is the most common method performed clinically in the United States, and several techniques exist. Recently, the use of infrared cameras has been explored by several groups. This study shows the use of this technology and its ability to assess arm volume measurements comparable to the Perometer while being more inexpensive, having a smaller footprint in clinics, and being easy to use. Methods and Results: Seventy-three BC survivors were scanned with the Perometer and the Kinect IR system, and arm volumes of 146 arms were calculated using their respective algorithms. Good correlation in the arm volume (R-squared = 0.8799) and percent difference (R-squared range 0.6277-0.7098) in the ranges where lymphedema diagnosis is made, and good clinical agreement of lymphedema status based on commonly used thresholds were found between the two modalities. The Cohen's kappa coefficient indicates that based on a 10% arm volume difference, there was a fair agreement (κ = 0.2663), and based on a 200 mL difference, there was a moderate agreement (κ = 0.5475) between the clinical outcomes of these two modalities. The Bland-Altman analysis of volume differences indicated a bias of 6.016 mL; thus, only a minor correction factor is necessary between the two modalities. Conclusion: The Kinect IR system presented can be used clinically for arm volume measurements to detect and monitor patients at risk for or managing lymphedema.
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Técnicas Biosensibles , Linfedema del Cáncer de Mama/diagnóstico , Supervivientes de Cáncer , Rayos Infrarrojos , Extremidad Superior/diagnóstico por imagen , Extremidad Superior/patología , Anciano , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Persona de Mediana EdadRESUMEN
Lymphatic Filariasis, a Neglected Tropical Disease, is caused by thread-like parasitic worms, including B. malayi, which migrate to the human lymphatic system following transmission. The parasites reside in collecting lymphatic vessels and lymph nodes for years, often resulting in lymphedema, elephantiasis or hydrocele. The mechanisms driving worm migration and retention within the lymphatics are currently unknown. We have developed an integrated in vitro imaging platform capable of quantifying B. malayi migration and behavior in a multicellular microenvironment relevant to the initial site of worm injection by incorporating the worm in a Polydimethylsiloxane (PDMS) microchannel in the presence of human dermal lymphatic endothelial cells (LECs) and human dermal fibroblasts (HDFs). The platform utilizes a motorized controllable microscope with CO2 and temperature regulation to allow for worm tracking experiments with high resolution over large length and time scales. Using post-acquisition algorithms, we quantified four parameters: 1) speed, 2) thrashing intensity, 3) percentage of time spent in a given cell region and 4) persistence ratio. We demonstrated the utility of our system by quantifying these parameters for L3 B. malayi in the presence of LECs and HDFs. Speed and thrashing increased in the presence of both cell types and were altered within minutes upon exposure to the anthelmintic drug, tetramisole. The worms displayed no targeted migration towards either cell type for the time course of this study (3 hours). When cells were not present in the chamber, worm thrashing correlated directly with worm speed. However, this correlation was lost in the presence of cells. The described platform provides the ability to further study B. malayi migration and behavior.