RESUMO
Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.
Assuntos
Escherichia coli/química , RNA Ribossômico 23S/química , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sítios de Ligação , Ácido Edético/metabolismo , Escherichia coli/genética , Compostos Ferrosos/metabolismo , Engenharia Genética , Radical Hidroxila/metabolismo , Modelos Moleculares , Sondas Moleculares/metabolismo , Peso Molecular , Mutação/genética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/genética , Ésteres do Ácido Sulfúrico/metabolismoRESUMO
Bryostatin 1 is currently in phase II clinical trial sponsored by the National Cancer Institute as an anticancer chemotherapeutic agent. Bryostatin 1 for injection was supplied in a dual pack containing a drug vial and a diluent vial and was manufactured by Ben Venue Laboratories, Inc (Bedford, OH). The stability and compatibility of the bryostatin 1-PET formulation, diluted to 1 and 10 ug/mL in saline and benzyl alcohol preserved saline, with polypropylene (PP) and polyvinyl chloride (PVC) bags at room temperature (27 degrees C) were studied. All experiments were conducted in triplicate and analyses were performed using a validated, stability-indicating, high performance liquid chromatography (HPLC) assay. Bryostatin 1 solutions were compatible with PP bags. At both concentrations and with both salines, the bryostatin content remained unchanged during the 28-day storage period, benzyl alcohol concentration in the preserved saline solutions also remained relatively constant. In PVC bags, however, a decrease in bryostatin 1 concentrations without generation of decomposition products was observed at both dilutions and with both salines during the 28-day storage. A decrease in benzyl alcohol concentration in the preserved saline was also observed. While no diethylhexylphthalate (DEHP) leakage into the solution was observed in PP bags, DEHP leakage in PVC infusion bags was observed on day 2 of storage which increased with storage time and leveled off on day 6. The amount of DEHP leached into drug solution is dependent on the drug concentration. This study suggests bryostatin-PET formulation diluted with preserved saline can be used for long-term (4 week) intravenous administration using PP infusion bags, but not with PVC bags.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/química , Lactonas/administração & dosagem , Lactonas/química , Briostatinas , Cromatografia Líquida de Alta Pressão , Dietilexilftalato , Estabilidade de Medicamentos , Infusões Intravenosas/instrumentação , Macrolídeos , Polipropilenos , Cloreto de Polivinila , SoluçõesRESUMO
Quinobene is the tetrasodium salt of an organic tetrasulphonic acid. Its unusual solubility characteristics makes the development of LC analysis difficult. However, a specific, precise and accurate LC assay was eventually achieved for quinobene. The assay required gradient elution and was not efficient for quinobene with respect to plate number. As an alternative, a capillary electrophoresis (CE) assay was also developed for quinobene. The CE assay was comparable to the LC assay in precision and accuracy. It was unaffected by the unusual solubility characteristics of quinobene and was more specific, efficient and rugged than the LC assay.
