RESUMEN
The development of effective strategies for gene therapy has been hampered by difficulties verifying transgene delivery in vivo and quantifying gene expression non-invasively. Magnetic resonance imaging (MRI) offers high spatial resolution and three-dimensional views, without tissue depth limitations. The iron-storage protein ferritin is a prototype MRI gene reporter. Ferritin forms a paramagnetic ferrihydrite core that can be detected by MRI via its effect on the local magnetic field experienced by water protons. In an effort to better characterize the ferritin reporter for central nervous system applications, we expressed ferritin in the mouse brain in vivo using a neurotropic herpes simplex virus type 1 (HSV-1). We computed three-dimensional maps of MRI transverse relaxation rates in the mouse brain with ascending doses of ferritin-expressing HSV-1. We established that the transverse relaxation rates correlate significantly to the number of inoculated infectious particles. Our results are potentially useful for quantitatively assessing limitations of ferritin reporters for gene therapy applications.
Asunto(s)
Encéfalo/diagnóstico por imagen , Ferritinas/aislamiento & purificación , Terapia Genética , Herpesvirus Humano 1/aislamiento & purificación , Imagen por Resonancia Magnética/métodos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ferritinas/química , Ferritinas/uso terapéutico , Expresión Génica , Regulación Viral de la Expresión Génica , Genes Reporteros , Herpesvirus Humano 1/genética , Ratones , RadiografíaRESUMEN
Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.
Asunto(s)
Anaplasma/crecimiento & desarrollo , Anaplasmosis/transmisión , Proteínas de la Membrana Bacteriana Externa/fisiología , Enfermedades de los Bovinos/transmisión , Dermacentor/microbiología , Infestaciones por Garrapatas/veterinaria , Anaplasma/genética , Anaplasma/ultraestructura , Anaplasmosis/parasitología , Animales , Adhesión Bacteriana/genética , Proteínas de la Membrana Bacteriana Externa/genética , Western Blotting/veterinaria , Bovinos , Enfermedades de los Bovinos/parasitología , ADN Bacteriano/genética , ADN Ribosómico/genética , Vectores de Enfermedades , Electroforesis en Gel de Poliacrilamida/veterinaria , Escherichia coli/genética , Femenino , Florida , Masculino , Oklahoma , Reacción en Cadena de la Polimerasa/veterinaria , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Glándulas Salivales/parasitología , Infestaciones por Garrapatas/microbiología , Infestaciones por Garrapatas/parasitologíaAsunto(s)
Medicina Clínica , Ortopedia , Investigadores , Internado y Residencia , Ortopedia/educaciónRESUMEN
The training of composite skills requiring differential responding to a large set of stimuli raises issues about how to break down the whole task into parts and which parts should be trained first. Components of Morse code reception skill were identified, separated, and used to test whether initial training on a difficult part was more effective than initial training on an easy part. Initial training on a difficult subset of stimuli and on a difficult subtask both yielded disadvantages rather than the advantage implied by recent findings with different tasks. Incremental training should begin with the part yielding the most effective strategic skills, which appear to depend on characteristics of the task. In both present experiments, easy initial training led to adoption of an effective unitization strategy for representing codes. The hypothesis that procedural reinstatement at delayed testing leads to better retention was supported and extended.
Asunto(s)
Percepción Auditiva , Retención en Psicología , Enseñanza , Humanos , Tiempo de Reacción , Factores de TiempoAsunto(s)
Ortopedia , Humanos , Ortopedia/estadística & datos numéricos , Estados Unidos , Recursos HumanosRESUMEN
What constitutes orthopaedic practice and how many orthopaedic surgeons are desirable for a given population has been discussed since the specialty was founded. The American Academy of Orthopaedic Surgeons began addressing this issue in January 1937. Extensive studies were done in the early 1970s with sponsorship from the American Academy of Orthopaedic Surgeons, the American College of Surgeons, the American Surgical Association, and the Division of Manpower Intelligence of the Department of Health Education and Welfare. These studies involved questionnaire surveys, Delphi panel modeling, and direct observation in a three-day time and motion study of a statistical sample of 150 practices. At the conclusion of these studies, it was observed that orthopaedics was largely a male specialty, practitioners preferred the surgical aspect of their practices to their office practices, that there was no type of practice that was more efficient than another, that the more orthopaedic surgeons there were in a population the more operative procedures were being done, and the character of the practice changes with the ratio of orthopaedist to population drops below 1 to 15,000. After 30 years involvement in health manpower issues the author concludes that there is no substitute for developing a solid database and analyzing trends, that the predictions have been remarkably accurate and although honorable men and women may disagree on the interpretation of data, few will argue that there is a limit on the number of orthopaedic procedures that can be justified in the diagnosis and treatment of a population. The essence of professionalism is self regulation and doing first and foremost what is in the best interest of the patient and society whether there necessitates an increase or a decrease in the number of orthopaedic surgeons being trained or practicing in a given population.
Asunto(s)
Ortopedia , Humanos , Procedimientos Ortopédicos/estadística & datos numéricos , Procedimientos Ortopédicos/tendencias , Estados Unidos , Recursos HumanosRESUMEN
The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.
Asunto(s)
Hormona Paratiroidea/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Receptores de Hormona Paratiroidea/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The crystal structures of four active site-directed thrombin inhibitors, 1-4, in a complex with human alpha-thrombin have been determined and refined at up to 2.0 A resolution using X-ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3-disubstituted benzo[b]thiophene structure. Compared to traditional active-site directed inhibitors, the X-ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C-3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C-2 was found to point away from the active site, occupying a location between the S1 and S1' sites. Together, the aromatic rings of the C-2 and C-3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence "Tyr-Pro-Pro-Trp." [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323-373).] In contrast to the binding mode of more classical thrombin inhibitors (D-Phe-Pro-Arg-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three-dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure-based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900-fold more potent than the screening hit 1.
Asunto(s)
Inhibidores Enzimáticos/química , Tiofenos/química , Trombina/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Relación Estructura-Actividad , Trombina/antagonistas & inhibidoresRESUMEN
Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.
Asunto(s)
Antitrombinas/síntesis química , Imidazoles/metabolismo , Péptidos/síntesis química , Antitrombinas/química , Antitrombinas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Modelos Moleculares , Péptidos/química , Péptidos/farmacologíaRESUMEN
Racial and cultural diversity in the physician workforce (in orthopaedics this includes women) is an essential if the best healthcare for the American people is to be provided. The percentage of minorities to the white population is increasing yet their representation in medical schools and the practicing community has not risen at that same pace. Affirmative Action efforts continue to be challenged as lowering standards and depriving better qualified students admission to medical schools. Admissions committees and orthopaedic residency directors should recognize that grade point averages and test scores may not be the best predictors of how well a physician or surgeon cares for patients. How far a person has come and what obstacles they have had to overcome, their demonstrated caring and compassion for people, their understanding of diverse cultures and languages may be a far better predictor of the quality of care physicians give to their patients. Affirmative Action should not be looked on as lowering standards, but using all available information in the selection process for medical school and residency training to ensure that the medical profession more closely mirrors the diverse racial and ethnic background of the United States population. How far a person has come and the adversity they have overcome may have far greater impact on making the correct diagnosis and setting out a proper treatment plan that the patient will accept than mastering test taking in the biologic and physical sciences. Obtaining racial and cultural diversity in the medical profession should be a national imperative.
Asunto(s)
Diversidad Cultural , Educación Médica , Grupos Minoritarios , Evaluación Educacional , Etnicidad , Femenino , Predicción , Humanos , Internado y Residencia , Masculino , Ortopedia/educación , Relaciones Médico-Paciente , Médicos , Calidad de la Atención de Salud , Grupos Raciales , Criterios de Admisión Escolar , Facultades de Medicina , Cambio Social , Estados Unidos , Población BlancaRESUMEN
Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.
Asunto(s)
Tiofenos/química , Trombina/antagonistas & inhibidores , Animales , Anticoagulantes/química , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Sitios de Unión , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Humanos , Modelos Moleculares , Ratas , Relación Estructura-Actividad , Tiofenos/farmacología , Tiofenos/uso terapéutico , Trombina/química , Trombosis/tratamiento farmacológicoRESUMEN
We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Raf-1. Specifically, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491-508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCalpha phosphorylation sites failed to significantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no effect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of different stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a significant mechanism of Raf-1 kinase regulation in COS-7 cells.
Asunto(s)
Factor de Crecimiento Epidérmico/fisiología , Regulación de la Expresión Génica , Oncogenes , Proteínas Proto-Oncogénicas c-raf/genética , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Guanosina Trifosfato/metabolismo , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de Aminoácido , Serina/metabolismo , Tirosina/metabolismo , Proteínas ras/fisiologíaRESUMEN
In an effort to increase the thrombin inhibitory activity of a novel series of inhibitors (i.e., 1a), substituents were incorporated at the C-3" position of the C-3 aryl ring (2). Consistent with the X-ray crystallography studies, small hydrophobic groups at the C-3" site (Br and Me) enhanced thrombin inhibitory activity by 8-fold. However, a few more hydrophilic substituents (NO2 and OMe) also enhanced the potency of the series. The biological results are discussed in terms of molecular modeling studies.
Asunto(s)
Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Tiofenos/química , Trombina/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Modelos Químicos , Modelos Moleculares , Relación Estructura-Actividad , Tiofenos/farmacologíaRESUMEN
Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki = 2 nM) and antiviral agent (HIV-1 ED50 = 14 nM). An X-ray cocrystal structure of the enzyme-AG1343 complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor interact with the S1 and S2 subsites of HIV-1 protease, respectively. In vivo studies indicate that AG1343 is well absorbed orally in a variety of species and possesses favorable pharmacokinetic properties in humans. AG1343 (Viracept) has recently been approved for marketing for the treatment of AIDS.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/enzimología , Nelfinavir/síntesis química , Nelfinavir/farmacología , Administración Oral , Animales , Fármacos Anti-VIH/farmacocinética , Disponibilidad Biológica , Callithrix , Perros , Relación Dosis-Respuesta a Droga , Femenino , Inhibidores de la Proteasa del VIH/farmacocinética , VIH-1/efectos de los fármacos , Macaca fascicularis , Masculino , Nelfinavir/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
The crystal structure of human alpha-thrombin in complex with LY178550, a nonpeptidyl, active site-directed inhibitor, has been solved to 2.07 A resolution by the method of X-ray crystallography. The final model of the complex has a crystallographic R-value of 21.5% (Rfree = 23.1%) with 0.014 A and 2.4 degrees standard deviation from ideal bond lengths and angles, respectively. Well-defined electron density was observed for the inhibitor in the active site. The inhibitor binds to the active site in an L-shaped manner, mimicking the bound conformation of the tripeptide arginal series of thrombin inhibitors (Chirgadze NY et al., 1992, American Crystallographic Association Meeting 20: 116 [Abstr. PB311]). The basic amidine of LY178550 forms a salt bridge with Asp 189 within the specificity pocket, while the 4-benzylpiperidine side chain engages in a number of hydrophobic interactions at the S2 and S3 binding sites. The inhibitor does not interact in any fashion with the active site sequence Ser 214-Gly 216, as occurs with many of the inhibitors studied previously. The indole N-H of the inhibitor forms a hydrogen bond to the gamma-oxygen of the catalytic serine (Ser 195).
Asunto(s)
Indoles/química , Piperidinas/química , Trombina/antagonistas & inhibidores , Trombina/química , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Hirudinas/análogos & derivados , Hirudinas/química , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Conformación ProteicaRESUMEN
In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes--together or individually--act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol, 1995; 19:1-9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10(-5) M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10(-5) M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes.