RESUMEN
The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.
Asunto(s)
Mejoramiento Genético/métodos , Mutagénesis Sitio-Dirigida/métodos , Recombinación Genética/genética , Sirolimus/metabolismo , Streptomyces/fisiología , Sirolimus/aislamiento & purificación , Especificidad de la Especie , Streptomyces/clasificaciónRESUMEN
Approximately one quarter of all hospitalized patients over age 75 years have a secondary diagnosis of dementia. A unique hospital-wide program to encourage appropriate communication techniques with patients who have dementia was provided to all departments of a hospital. Evaluation indicated improvement in some communication techniques. Additional education is needed to disperse the information to as many staff as possible and to sustain the change.
Asunto(s)
Competencia Clínica , Comunicación , Demencia/enfermería , Relaciones Enfermero-Paciente , Desarrollo de Personal/métodos , Factores de Edad , Anciano , Distribución de Chi-Cuadrado , Escolaridad , Humanos , Modelos Organizacionales , Innovación Organizacional , Proyectos Piloto , Desarrollo de Programa/métodos , Evaluación de Programas y Proyectos de SaludRESUMEN
Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 µM and 0.16 µM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F<4%) and low solubility (<25 µM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2>5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.
Asunto(s)
Antivirales/farmacología , Ciclofilinas/antagonistas & inhibidores , Lactonas/farmacología , Animales , Antivirales/química , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Células Hep G2 , Hepacivirus/efectos de los fármacos , Humanos , Lactonas/química , Masculino , Ratones , Estructura Molecular , Replicación Viral/efectos de los fármacosRESUMEN
The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.
Asunto(s)
Proteínas Bacterianas , Ácido Corísmico/metabolismo , Genes Bacterianos/fisiología , Inmunosupresores/metabolismo , Familia de Multigenes/fisiología , Sirolimus/metabolismo , Streptomyces , Tacrolimus/análogos & derivados , Tacrolimus/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Ácido Corísmico/química , Inmunosupresores/química , Sirolimus/química , Streptomyces/enzimología , Streptomyces/genética , Tacrolimus/químicaRESUMEN
As the population ages, the number of patients with dementia will increase rapidly. Nurses caring for patients with dementia will need knowledge of interventions for communicating; managing agitation, nutrition, hygiene, and pain; providing spiritual care; and determining decision-making capacity.
Asunto(s)
Demencia , Necesidades y Demandas de Servicios de Salud/organización & administración , Pacientes Internos , Anciano , Trastornos de la Comunicación/etiología , Demencia/complicaciones , Demencia/epidemiología , Demencia/enfermería , Evaluación Geriátrica , Enfermería Geriátrica/organización & administración , Humanos , Higiene , Actividades Recreativas , Competencia Mental , Rol de la Enfermera , Evaluación en Enfermería , Apoyo Nutricional , Dolor/etiología , Agitación Psicomotora/etiología , EspiritualidadRESUMEN
A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.
Asunto(s)
Benzoquinonas/farmacología , Productos Biológicos/farmacología , Ingeniería Genética , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Benzoquinonas/química , Benzoquinonas/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
Studies indicate 40% of indwelling urinary catheters are unnecessary in hospitalized patients (Gardam, Amihod, Orenstein, Consolacion, & Miller, 1998; Gokula, Hickner, & Smith, 2004). The results of a protocol developed to limit catheter use are described.