Mutation of Ser172 in Yeast β Tubulin Induces Defects in Microtubule Dynamics and Cell Division

Ser172 of β tubulin is an important residue that is mutated in a human brain disease and phosphorylated by the cyclin-dependent kinase Cdk1 in mammalian cells. To examine the role of this residue, we used the yeast S. cerevisiae as a model and produced two different mutations (S172A and S172E) of the conserved Ser172 in the yeast β tubulin Tub2p. The two mutants showed impaired cell growth on benomyl-containing medium and at cold temperatures, altered microtubule (MT) dynamics, and altered nucleus positioning and segregation. When cytoplasmic MT effectors Dyn1p or Kar9p were deleted in S172A and S172E mutants, cells were viable but presented increased ploidy. Furthermore, the two β tubulin mutations exhibited synthetic lethal interactions with Bik1p, Bim1p or Kar3p, which are effectors of cytoplasmic and spindle MTs. In the absence of Mad2p-dependent spindle checkpoint, both mutations are deleterious. These findings show the importance of Ser172 for the correct function of both cytoplasmic and spindle MTs and for normal cell division

Residual solvent determination by head space gas chromatography with flame ionization detector in omeprazole API

Residual solvents in pharmaceutical samples are monitored using gas chromatography with head space. Based on good manufacturing practices, measuring residual solvents is mandatory for the release testing of all active pharmaceutical ingredients (API). The analysis of residual organic solvents (methanol, acetone, cyclohexane, dichloromethane, toluene) in Omeprazole, an active pharmaceutical ingredient was investigated. Omeprazole is a potent reversible inhibitor of the gastric proton pump H+/K+-ATPase. The Head space gas chromatography (HSGC) method described in this investigation utilized a SPB TM-624, Supelco, 30 m long x 0.25 mm internal diameter, 1.4µm-thick column. Since Omeprazole is a thermally labile compound, the selection of the proper injector temperature is critical to the success of the analysis. The injector temperature was set at 170ºC to prevent degradation. The initial oven temperature was set at 40ºC for 12 min and programmed at a rate of 10ºC min-1 to a final temperature of 220ºC for 5 min. Nitrogen was used as a carrier gas. The sample solvent selected was N,N-dimethylacetamide. The method was validated to be specific, linear, precise, sensitive, rugged and showed excellent recovery.<br>Solventes residuais em amostras farmacêuticas são monitoradas utilizando-se cromatografia a gás "headspace". Com base nas boas práticas de fabricação, a medida de solventes residuais é obrigatória para o teste de liberação de todos os ingredientes farmacêuticos (API). Efetuou-se a análise de solventes orgânicos residuais (metanol, acetona, cicloexano, diclorometano, tolueno) em omeprazol, ingrediente farmacêutico ativo. O omeprazol é potente inibidor reversível da bomba de prótons H+/K+-ATPase. A cromatografia a gás "headspace" (HSGC) descrita nessa pesquisa utilizou um SPB TM-624, Supelco, de 30 m de comprimento x 0,25 mm de diâmetro interno, e coluna de 1,4 µm de espessura. Considerando-se que o omeprazol é termicamente lábil, a seleção da temperatura apropriada do injetor é crítica para impedir a degradação. A temperatura inicial do forno foi de 40 ºC, por 12 minutos, e programada à taxa de acréscimo de 10 ºC min-1 até a temperatura final de 220 ºC, por 5 minutos. Nitrogênio foi utilizado como gás de transporte. Selecionou-se como solvente a N,N-dimetilacetamida. O método foi validado mostrando-se específico, linear, preciso, sensível, robusto e com excelente recuperação