RESUMEN
A walking gait has been identified in a range of vertebrate species with different body plans, habitats, and life histories. With increased application of this broad umbrella term, it has become necessary to assess the physical characteristics, analytical approaches, definitions, and diction used to describe walks. To do this, we reviewed studies of slow speed locomotion across a range of vertebrates to refine the parameters used to define walking, evaluate analytical techniques, and propose approaches to maximize consistency across subdisciplines. We summarize nine key parameters used to characterize walking behaviors in mammals, birds, reptiles, amphibians, and fishes. After identifying consistent patterns across groups, we propose a comprehensive definition for a walking gait. A walk is a form of locomotion where the majority of the forward propulsion of the animal comes from forces generated by the appendages interacting with the ground. During a walk, an appendage must be out of phase with the opposing limb in the same girdle and there is always at least one limb acting as ground-support (no suspension phase). Additionally, walking occurs at dimensionless speeds <1 v* and the duty factor of the limbs is always >0.5. Relative to other gaits used by the same species, the stance duration of a walk is long, the cycle frequency is low, and the cycle distance is small. Unfortunately, some of these biomechanical parameters, while effectively describing walks, may also characterize other, non-walking gaits. Inconsistent methodology likely contributes to difficulties in comparing data across many groups of animals; consistent application of data collection and analytical techniques in research methodology can improve these comparisons. Finally, we note that the kinetics of quadrupedal movements are still poorly understood and much work remains to be done to understand the movements of small, exothermic tetrapods.
RESUMEN
The migration, adhesion, and subsequent extravasation of leukocytes into inflamed tissues contribute to the pathogenesis of a variety of inflammatory diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. The integrin adhesion receptor alpha 4 beta 1 expressed on leukocytes binds to the extracellular matrix protein fibronectin and to the cytokine inducible vascular cell adhesion molecule-1 (VCAM-1) at inflamed sites. Binding of alpha 4 beta 1 to VCAM-1 initiates firm adhesion of the leukocyte to the vascular endothelium followed by extravasation into the tissue. Monoclonal antibodies generated against either alpha 4 beta 1 or VCAM-1 can moderate this inflammatory response in a variety of animal models. Recently peptides containing a consensus LDV sequence based on the connecting segment-1 (CS-1) of fibronectin and cyclic peptides containing an RCD motif have shown promise in modulating leukocyte migration and inflammation presumably by blocking the interaction of alpha 4 beta 1 with VCAM-1. Here we describe novel, highly potent, cyclic peptides that competitively inhibit alpha 4 beta 1 binding to VCAM-1 and fibronectin at sub nanomolar concentrations. The structure of a representative analog was determined via NMR spectroscopy and used to facilitate optimization of peptide leads. The peptides discussed here utilize similar functional groups as the binding epitope of VCAM-1, inhibit lymphocyte migration in vivo, and are highly selective for alpha 4 beta 1. Furthermore the structure--activity relationships described here have provided a template for the structure-based design of small molecule antagonists of alpha 4 beta 1-mediated cell adhesion processes.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Integrinas/antagonistas & inhibidores , Linfocitos/fisiología , Péptidos Cíclicos/síntesis química , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Integrina alfa4beta1 , Integrinas/inmunología , Integrinas/metabolismo , Linfocitos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Modelos Moleculares , Estructura Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Receptores Mensajeros de Linfocitos/inmunología , Receptores Mensajeros de Linfocitos/metabolismo , Relación Estructura-Actividad , Molécula 1 de Adhesión Celular Vascular/química , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
A structural survey of protein Zn2+ binding geometries was instigated based upon the functional requirement of Ras farnesyltransferase for Zn2+. The Cys-X-X-Cys motif found in Zn(2+)-binding proteins such as aspartate transcarbamylase was used as a template to devise a bidentate-coordination model for Cys-A1-A2-X peptide inhibitors. Accordingly, replacement of the central dipeptide with the hydrophobic scaffold 3-amino-1-carboxymethyl-2,3-dihydro-5- phenyl-1H-1,4-benzodiazepin-2-one (BZA) yielded a peptidomimetic inhibitor, Cys(BZA)Met, of moderate potency (IC50 = 400 nM). N-Methylation of the cysteine amide improved potency almost 100-fold (IC50 = 0.3-1 nM). The increased affinity presumably correlates with a preferred conformation of the inhibitor which maximizes a hydrophobic interaction between the scaffold and the enzyme, and the proper presentation of cysteine and methionine to allow bidentate coordination at Zn2+. These non-peptide inhibitors have been shown to block farnesylation of the Ras protein in intact cells and provide lead compounds for the development of new cancer therapeutic agents.
Asunto(s)
Transferasas Alquil y Aril , Benzodiazepinas/farmacología , Transferasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Benzodiazepinas/síntesis química , Permeabilidad de la Membrana Celular , Farnesiltransferasa , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Relación Estructura-Actividad , Transferasas/metabolismoRESUMEN
Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable enterotoxin receptor-guanylyl cyclase. Guanylin is synthesized as a 115-amino acid prohormone, proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s). In order to examine the processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli. The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion. Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized. Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity. Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure.
Asunto(s)
Hormonas Gastrointestinales , Biosíntesis de Péptidos , Precursores de Proteínas/biosíntesis , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Clonación Molecular/métodos , GMP Cíclico/metabolismo , ADN Complementario/metabolismo , Escherichia coli , Expresión Génica , Guanilato Ciclasa/metabolismo , Humanos , Intestino Delgado/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos Natriuréticos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Péptidos/aislamiento & purificación , Péptidos/farmacología , Pliegue de Proteína , Precursores de Proteínas/aislamiento & purificación , Precursores de Proteínas/farmacología , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Mapeo Restrictivo , TripsinaRESUMEN
A solid-phase approach using trinucleotide building blocks and functionalized cellulose as solid support was used to synthesize the 22 oligodeoxynucleotides that constitute a totally synthetic gene coding for a 43 amino acid peptide. Gastric inhibitory Polypeptide. The overall strategy used for the design and synthesis of the oligomers is described.