Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Defining Sales and Operations Planning Process in an Environmental Management Company

Sales and operations research publications have increased significantly in the last decades. The concept of sales and operations planning (S&OP) has gained increased recognition and has been put forward as the area within Supply Chain Management (SCM). Development of S&OP is based on the need for determining future actions, both for sales and operations, since off-shoring, outsourcing, complex supply chains and extended lead times make challenges for responding to changes in the marketplace when they occur. Order intake of the case company has grown rapidly during the last years. Along with the growth, new challenges considering data management and information flow have arisen due to increasing customer orders. To manage these challenges, case company has implemented S&OP process, though initial process is in early stage and due to this, the process is not managing the increased customer orders adequately. Thesis objective is to explore extensively the S&OP process content of the case company and give further recommendations. Objectives are categorized into six different groups, to clarify the purpose of this thesis. Qualitative research methods used are active participant observation, qualitative interviews, enquiry, education, and a workshop. It is notable that demand planning was felt as cumbersome, so it is typically the biggest challenge in S&OP process. More proactive the sales forecasting can be, more expanded the time horizon of operational planning will turn out. S&OP process is 60 percent change management, 30 percent process development and 10 percent technology. The change management and continuous improvement can sometimes be arduous and set as secondary. It is important that different people are required to improve the process and the process is constantly evaluated. As well as, process governance is substantially in a central role and it has to be managed consciously. Generally, S&OP process was seen important and all the stakeholders were committed to the process. Particular sections were experienced more important than others, depending on the stakeholders’ point of views. Recommendations to objective groups are evaluated by the achievable benefit and resource requirement. The urgent and easily implemented improvement recommendations should be executed firstly. Next steps are to develop more coherent process structure and refine cost awareness. Afterwards demand planning, supply planning, and reporting should be developed more profoundly. For last, information technology system should be implemented to support the process phases.

Utilização do Intron Splice Site primer EI-1 na discriminação de leveduras contaminantes do processo de fermentação alcoólica

Neste trabalho, utilizamos o Intron Splice Site primer EI-1 para a análise do perfil de amplificação de diferentes espécies de leveduras consideradas contaminantes no processo de fermentação alcoólica, originadas de uma destilaria no Estado da Paraíba na safra 2004/2005. Foram realizadas as etapas analíticas para discriminação molecular das leveduras a partir da extração do DNA total, amplificação por PCR e análise do perfil genético. Os resultados obtidos indicam que o Intron Splice Site primer EI-1é muito eficaz na discriminação das diferentes espécies de Saccharomyces e não Saccharomyces, evidenciando padrões de bandas específicos para as leveduras analisadas. Este primer, por ser complementar a uma região muito conservada do genoma das leveduras, mostrou-se incapaz de uma discriminação intraespecífica. Isto demonstra a utilidade deste marcador no auxílio à taxonomia de leveduras.