ABSTRACT
The current chapter focuses on the use of filamentous phages to display and modify biologically active cytokines, with special emphasis on directed evolution of novel variants showing improved receptor binding. Cytokines are essential protein mediators involved in cell-to-cell communication. Their functional importance and the complexity of their interactions with multichain receptors make cytokine engineering a promising tool for the discovery and optimization of therapeutic molecules. Protocols used at the laboratory are illustrated through examples of manipulation of interleukin-2 and interleukin-6, two members of the family of alpha-helix-bundle cytokines playing pivotal roles in immunity and inflammation.
Subject(s)
Bacteriophages , Cytokines , Humans , Interleukin-6 , Cell Communication , InflammationABSTRACT
Animals raised in captivity during several generations may not express appropriate antipredator behaviour when reintroduced into the wild. Here we present the results of experiments to enhance behavioural responses to predators in adult males of the endangered lizard Gallotia simonyi (El Hierro, Canary Islands). Individuals were subjected to a training procedure (control, pre-training, training and post-training phases) using stuffed specimens of a kestrel and a cat as predators. We filmed all trials and compared relative durations of the more common behaviour patterns shown by lizards, both among experimental phases and before and after presentation of the stuffed predator. Locomotion and Basking were significantly reduced in the training and post-training trials and also after stimulus presentation, suggesting that the training protocol induced lizard avoidance over both predator models. To our knowledge, this is the first time lizards have been trained to show antipredator avoidance and our results provide the basis for a new management strategy that could be useful for reintroduction of captive-bred individuals of endangered species.
Subject(s)
Avoidance Learning , Behavior, Animal , Endangered Species , Animals , Breeding , Lizards , Male , SpainABSTRACT
We report the development of a potent, selective histone deacetylase 6 (HDAC6) inhibitor. This HDAC6 inhibitor blocks growth of normal and transformed cells but does not induce death of normal cells. The HDAC6 inhibitor alone is as effective as paclitaxel in anticancer activity in tumor-bearing mice.
Subject(s)
Antineoplastic Agents/chemical synthesis , Benzeneacetamides/chemistry , Benzeneacetamides/pharmacology , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylases/genetics , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Acetylation/drug effects , Animals , Antineoplastic Agents/chemistry , Cell Death/drug effects , Cell Proliferation/drug effects , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/chemistry , Mice , Molecular Structure , Paclitaxel , Tubulin/metabolismABSTRACT
Development of isoform-selective histone deacetylase (HDAC) inhibitors is important in elucidating the function of individual HDAC enzymes and their potential as therapeutic agents. Among the eleven zinc-dependent HDACs in humans, HDAC6 is structurally and functionally unique. Here, we show that a hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalytic activity in vivo and in vitro. HPOB causes growth inhibition of normal and transformed cells but does not induce cell death. HPOB enhances the effectiveness of DNA-damaging anticancer drugs in transformed cells but not normal cells. HPOB does not block the ubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitor HPOB has therapeutic potential in combination therapy to enhance the potency of anticancer drugs.
Subject(s)
Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Acetylation/drug effects , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Cell Line, Transformed/drug effects , Cell Line, Tumor , Cell Survival/drug effects , DNA Breaks, Double-Stranded/drug effects , Doxorubicin/pharmacology , Etoposide/pharmacology , Histone Deacetylase Inhibitors/chemistry , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Mice , Trehalose/pharmacology , Tubulin/metabolism , Vorinostat , Xenograft Model Antitumor AssaysABSTRACT
Histone deacetylase inhibitors (HDACi) are a new group of anticancer drugs with tumor selective toxicity. Normal cells are relatively resistant to HDACi-induced cell death compared with cancer cells. Previously, we found that vorinostat induces DNA breaks in normal and transformed cells, which normal but not cancer cells can repair. In this study, we found that checkpoint kinase 1 (Chk1), a component of the G2 DNA damage checkpoint, is important in the resistance of normal cells to HDACi in vitro and in vivo. Inhibition of Chk1 activity with Chk1 inhibitor (UCN-01, AZD7762, or CHIR-124) in normal cells increases their sensitivity to HDACi (vorinostat, romidepsin, or entinostat) induced cell death, associated with extensive mitotic disruption. Mitotic abnormalities included loss of sister chromatid cohesion and chromosomal disruption. Inhibition of Chk1 did increase HDACi-induced cell death of transformed cells. Thus, Chk1 is an important factor in the resistance of normal cells, and some transformed cells, to HDACi-induced cell death. Use of Chk1 inhibitors in combination with anticancer agents to treat cancers may be associated with substantial toxicity.
Subject(s)
Drug Resistance , Histone Deacetylase Inhibitors/pharmacology , Protein Kinases/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Checkpoint Kinase 1 , DNA Damage , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Foreskin/cytology , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Immunoblotting , Male , Mice , Protein Kinases/genetics , Quinolines/pharmacology , Quinuclidines/pharmacology , RNA Interference/physiology , Spleen/drug effects , Spleen/metabolism , Staurosporine/analogs & derivatives , Staurosporine/pharmacology , Thiophenes/pharmacology , Urea/analogs & derivatives , Urea/pharmacology , VorinostatABSTRACT
Histone deacetylase 6 (HDAC6) is structurally and functionally unique among the 11 human zinc-dependent histone deacetylases. Here we show that chemical inhibition with the HDAC6-selective inhibitor tubacin significantly enhances cell death induced by the topoisomerase II inhibitors etoposide and doxorubicin and the pan-HDAC inhibitor SAHA (vorinostat) in transformed cells (LNCaP, MCF-7), an effect not observed in normal cells (human foreskin fibroblast cells). The inactive analogue of tubacin, nil-tubacin, does not sensitize transformed cells to these anticancer agents. Further, we show that down-regulation of HDAC6 expression by shRNA in LNCaP cells enhances cell death induced by etoposide, doxorubicin, and SAHA. Tubacin in combination with SAHA or etoposide is more potent than either drug alone in activating the intrinsic apoptotic pathway in transformed cells, as evidenced by an increase in PARP cleavage and partial inhibition of this effect by the pan-caspase inhibitor Z-VAD-fmk. HDAC6 inhibition with tubacin induces the accumulation of γH2AX, an early marker of DNA double-strand breaks. Tubacin enhances DNA damage induced by etoposide or SAHA as indicated by increased accumulation of γH2AX and activation of the checkpoint kinase Chk2. Tubacin induces the expression of DDIT3 (CHOP/GADD153), a transcription factor up-regulated in response to cellular stress. DDIT3 induction is further increased when tubacin is combined with SAHA. These findings point to mechanisms by which HDAC6-selective inhibition can enhance the efficacy of certain anti-cancer agents in transformed cells.
Subject(s)
Anilides/pharmacology , Antineoplastic Agents/pharmacology , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/pathology , DNA Damage , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Caspases/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Checkpoint Kinase 2 , DNA Replication/drug effects , Down-Regulation/drug effects , Doxorubicin/pharmacology , Drug Screening Assays, Antitumor , Drug Synergism , Etoposide/pharmacology , G1 Phase/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase 6 , Histones/metabolism , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Topoisomerase II Inhibitors/pharmacology , Up-Regulation/drug effectsABSTRACT
There are 18 histone deacetylases (HDAC) generally divided into four classes based on homology to yeast HDACs. HDACs have many protein substrates in addition to histones that are involved in regulation of gene expression, cell proliferation, and cell death. Inhibition of HDACs can cause accumulation of acetylated forms of these proteins, thus altering their function. HDAC inhibitors (HDACi), such as the hydroxamic acid-based vorinostat (suberoylanilide hydroxamic acid), inhibit the zinc-containing classes I, II, and IV, but not the NAD(+)-dependent class III, enzymes. HDACis are a group of novel anticancer agents. Vorinostat is the first HDACi approved for clinical use in the treatment of the cancer cutaneous T-cell lymphoma. Factors affecting expression of HDACs are not well understood. This study focuses on the effect of the HDACi vorinostat on the expression of class I and class II HDACs. We found that vorinostat selectively down-regulates HDAC7 with little or no effect on the expression of other class I or class II HDACs. Fourteen cell lines were examined, including normal, immortalized, genetically transformed, and human cancer-derived cell lines. Down-regulation of HDAC7 by vorinostat is more pronounced in transformed cells sensitive to inhibitor-induced cell death than in normal cells or cancer cells resistant to induced cell death. Modulation of HDAC7 levels by small interfering RNA-mediated knockdown or by HDAC7 overexpression is associated with growth arrest but without detectable changes in acetylation of histones or p21 gene expression. Selective down-regulation of HDAC7 protein may serve as a marker of response of tumors to HDACi.
Subject(s)
Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Acetylation , Blotting, Northern , Blotting, Western , Cell Transformation, Neoplastic/pathology , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Fibroblasts/drug effects , Fibroblasts/enzymology , Histone Deacetylase Inhibitors , Histone Deacetylases/classification , Histone Deacetylases/genetics , Humans , Lung/drug effects , Lung/enzymology , Male , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/pathology , Prostate/drug effects , Prostate/enzymology , RNA, Small Interfering/pharmacology , Skin/drug effects , Skin/enzymology , VorinostatABSTRACT
There is a great need to develop better mechanism-based therapies for prostate cancer. In this investigation, we studied four human prostate cancer cell lines, LNCaP, DU145, LAPC4, and PC3, which differ in response to the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (vorinostat), a new anticancer drug. Examining the role of intrinsic mitochondrial caspase-dependent apoptosis and caspase-independent, reactive oxygen species (ROS) facilitated cell death, has provided an understanding of mechanisms that may determine the varied response to the histone deacetylase inhibitor. We found striking differences among these cancer cells in constitutive expression and response to suberoylanilide hydroxamic acid in levels of antiapoptotic and proapoptotic proteins, mitochondria membrane integrity, activation of caspases, ROS accumulation, and expression of thioredoxin, the major scavenger of ROS. Identifying these differences can have predictive value in assessing therapeutic response and identifying targets to enhance therapeutic efficacy.
Subject(s)
Antineoplastic Agents/metabolism , Apoptosis/physiology , Histone Deacetylase Inhibitors , Hydroxamic Acids/metabolism , Prostatic Neoplasms/metabolism , Thioredoxins/metabolism , Antineoplastic Agents/therapeutic use , Apoptosis Regulatory Proteins , Carrier Proteins/metabolism , Caspase Inhibitors , Caspases/metabolism , Cell Line, Tumor , Humans , Hydroxamic Acids/therapeutic use , Inhibitor of Apoptosis Proteins , Intracellular Signaling Peptides and Proteins/metabolism , Male , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Neoplasm Proteins/metabolism , Prostatic Neoplasms/drug therapy , Reactive Oxygen Species/metabolism , Survivin , Vorinostat , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Histone deacetylase (HDAC) inhibitors can induce various transformed cells to undergo growth arrest and/or death. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor which is in phase I/II clinical trials and has shown antitumor activity in hematologic and solid tumors at doses well tolerated by patients. HDAC is the target for SAHA, but the mechanisms of the consequent induced death of transformed cells are not completely understood. In this study, we report that SAHA induced polyploidy in human colon cancer cell line HCT116 and human breast cancer cell lines, MCF-7, MDA-MB-231, and MBA-MD-468, but not in normal human embryonic fibroblast SW-38 and normal mouse embryonic fibroblasts. The polyploid cells lost the capacity for proliferation and committed to senescence. The induction of polyploidy was more marked in HCT116 p21WAF1-/- or HCT116 p53-/- cells than in wild-type HCT116. The development of senescence of SAHA-induced polyploidy cells was similar in all colon cell lines. The present findings indicate that the HDAC inhibitor could exert antitumor effects by inducing polyploidy, and this effect is more marked in transformed cells with nonfunctioning p21WAF1 or p53 genes.
Subject(s)
Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Polyploidy , Cell Death/drug effects , Cell Death/genetics , Cell Growth Processes/drug effects , Cell Growth Processes/genetics , Colonic Neoplasms/enzymology , DNA Replication/drug effects , HCT116 Cells , Humans , VorinostatABSTRACT
Se realizó un estudio descriptivo para conocer el comportamiento de la mortalidad en menores de 5 años en el municipio Manzanillo desde 1989 a 1996. Se estudió el total de fallecidos distribuidos según grupos de edad y principales causas de muerte. La información se obtuvo de los registros de estadística continua y proyecciones de la población. Se calcularon las tasas específicas de mortalidad, índice de supervivencia y tasas de reducción media para establecer las diferencias entre los períodos a comparar. Se constató un descenso en la mortalidad en todos los grupos de edad. En menores de 1 año contribuyeron a la disminución las anomalías congénitas, los accidentes y las afecciones perinatales; en menores de 5 años las causas del descenso de este indicador fueron las anomalías congénitas y los accidentes. Se concluye que existe disminución de la mortalidad en el período de estudio con incremento del índice de supervivencia y se logró una reducción de la mortalidad respecto al período inicial de la investigación.(AU)
Subject(s)
Humans , Male , Female , Child , Infant Mortality , Survival Rate , Cause of Death , MortalityABSTRACT
En el presente estudio evaluamos el efecto de la metoclopramida intravenosa sobre la presión sanguínea en sujetos normotensos (no entrenados, futbolistas, corredores) y en pacientes hipertensos. Hubo una disminución de la presión arterial sólo observado en sujetos femeninos no entrenados y esta fue más grande en sujetos hipertensos. En futbolistas y corredores, la disminución en la presión arterial fue estadísticamente no significativa. Hubo un efecto no significativo sobre la frecuencia cardíaca. El mecanismo probable de este nuevo efecto farmacológico de la metoclopramida es desconocido; sin embargo, actualmente se están realizando investigaciones para definir su mecanismo de acción
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Blood Pressure , Hypertension , Metoclopramide/therapeutic use , Receptors, DopamineABSTRACT
Veinte (20) sujetos normotensos no entrenados y treinta y dos (32) sujetos normotensos entrenados (futbolistas y corredores) fueron estudiados en la Unidad de Farmacología Clínica de la Escuela de Medicina Vargas en Caracas, Venezuela. Los sujetos recibieron una infisión de solución de glucosa al 5 por ciento antes y despues de una infusión de Metoclopramida intravenosa a una dosis de 7,5 ug/kg/min por un período de 30 minutos. Los sujetos normotensos no entrenados respondieron con un aumento de la presión arterial después de la Prueba por Frío (PPF) (17,2 versus 21,4 mmHg en sistólica y 17,9 versus 24,1 mmHg en diastólica) antes y despues de la administración de Metoclopramida. Los futbolistas respondieron con un aumento de la presión arterial después de la PPF, pero sin embargo, la respuesta de la presión sistólica fue comparativamente menor. Los corredores respondieron con un aumento de la presión arterial después de la PPF (18,7 mmHg en sistólica y 15,8 versus 13,9 mmHd en diastólica) antes y después de la administración de Metoclopramida. La frecuencia cardíaca sólo aumentó en los sujetos entrenados antes de la Metoclopramida. Concluimos lo siguiente: 1. La hiperreactividad vascular inducida por la Metoclopramida está ausente en los sujetos entrenados. 2. La respuesta cardiovascular a la Prueba Presora por Frío está atenuada en los sujetos atletas en comparación con aquellos sujetos no entrenados. Esto podría significar una probable adaptación del sistema dopaminérgico durante el ejercicio
Subject(s)
Humans , Male , Female , Adult , Hypertension/diagnosis , Metoclopramide/therapeutic use , Pressoreceptors , Sports Medicine/trends , Caloric TestsABSTRACT
Cardiac workload was determined in normotensive and hypertensive subjects during submaximal treadmil exercise under metoclopramide treatment at the José Gregorio Hernández Hospital and at the Vargas Hospital, Caracas, Venezuela. Subjects were submitted to treadmil exercise during a 30 min period of 5 por ciento glucose solution administration (before and after metoclopramide) and during a 30 min period of metoclopramide, 7.5 µg/Kg/min administration. Treadmill exercise induced an increase of cardiac Workload in both normotensive and hypertensive subjects. Metoclopramide induced a decrease of cardiac workload in normotensives and less in hypertensives. We conclude that metoclopramide, a known DA2 dopaminergic blocker, reduces cardiac workload during keadmill exercise in normotensive and hypertensive subjects
Subject(s)
Humans , Male , Female , Cardiology , Exercise/physiology , Glucose/administration & dosage , Hypertension , Metoclopramide/therapeutic useABSTRACT
La pérdida perioperatoria excesiva de sangre ha sido siempre una complicación que pone en peligro de vida. Los médicos han intensificado los esfuerzos para minimizar las necesidades de productos sanguíneos, al aceptar un volumen globular más bajo para pacientes quirúrgicos. La hemodilución normovolémica aguda (HNA) consiste en la dilución del volumen eritrocítico (anemia dilucional) reemplazada por un sustituto libre de células, para mantener un volumen intravascular cercano a lo normal; pero sometiendo al sistema cardiovascular a adaptaciones para poder mantener su homeostasis. En esta revisión se considerarán los mecanismos y sus implicancias fisiológicas. Además se analizarán los distintos sustitutos y sus propiedades.
Subject(s)
Humans , Blood Loss, Surgical , Blood Transfusion , Cardiovascular System/physiology , Hemodilution , Oxygen Consumption , Postoperative Hemorrhage , Colloids/adverse effects , Colloids/pharmacokinetics , Monitoring, IntraoperativeABSTRACT
La pérdida perioperatoria excesiva de sangre ha sido siempre una complicación que pone en peligro de vida. Los médicos han intensificado los esfuerzos para minimizar las necesidades de productos sanguíneos, al aceptar un volumen globular más bajo para pacientes quirúrgicos. La hemodilución normovolémica aguda (HNA) consiste en la dilución del volumen eritrocítico (anemia dilucional) reemplazada por un sustituto libre de células, para mantener un volumen intravascular cercano a lo normal; pero sometiendo al sistema cardiovascular a adaptaciones para poder mantener su homeostasis. En esta revisión se considerarán los mecanismos y sus implicancias fisiológicas. Además se analizarán los distintos sustitutos y sus propiedades. (AU)
Subject(s)
Humans , Oxygen Consumption , Postoperative Hemorrhage , Blood Loss, Surgical , Hemodilution/statistics & numerical data , Cardiovascular System/physiology , Blood Transfusion , Monitoring, Intraoperative , Colloids/pharmacokinetics , Colloids/adverse effectsABSTRACT
Se describen y analizan 19 casos de Endocarditis Infecciosa diagnósticados y tratados entre 1989 y 1993 en el Hospital Central de Valencia y en el Hospital Policlínico Valencia. El 84,2 por ciento de los casos presentaban antecedentes de hospitalización prolongada en las Unidad de Cuidados Intensivos, o de uso de vía endovenoso central. En dos de los casos se demostró una vía endovenosa periférica como fuente de infección, y dos más se infectaron a partir de derivaciones ventrículo cava. Staphylococccus epidermidis fue el agente más frecuente identificado, seguido por Staphylococcus aureus y Pseudomonas aeruginosas. La valvula más frecuentemente afectada fue la tricúspide. La manifestación clínica que más frecuentemente motivó la consulta fue fiebre prolongada. El diagnóstico se realizó en base a los hallazgos clínicos, ecocardiográficos y microbiológicos (Hemocultivos seriados). La complicación más frecuente fue neumonía, aislandose del esputo la misma especie bacteriana que de los hemocultivos. La Endocarditis de cavidades derechas es una entidad a tener presente en los pacientes que han requerido uso de los cateteres endovenosos. El razonamiento clínico, la ecocardiografía y la bacteriológia juegan un rol de primera línea en el diagnóstico definitivo
