Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42

Studies on the catalytic acid/base residues of glutamate racemase

Glutamate racemase from Lactobacillus fermenti (murl, E.C. 5.1.1.3) is a cofactorindependent enzyme which catalyzes the interconversion of the enantiomers of glutamate using Cys73 and Cysl84 as the general acid/base catalysts. A cysteine thiolate abstracts the α-proton from one face of glutamate and the other cysteine thiol delivers a proton to the opposite face. The roles of the cysteine residues are explored using the two glutamate racemase mutant enzymes, C73S and C184S. The mutants retain the ability to racemize glutamate with specificity constants ~10³-fold lower than those of wild type enzyme. The mutant-catalyzed dehydration reaction of iV-hydroxyglutamate, a one-base requiring reaction, is used to determine which cysteine residue acts on each enantiomer of glutamate. With [sub D]-N-hydroxyglutamate, the C73S mutant is a poorer catalyst than the wild type enzyme, whereas theC184S mutant is a better catalyst. The opposite trend is observed with L-A^-hydroxyglutamate. The results suggest Cys73 is the residue responsible for the deprotonation of D-glutamate and Cysl84 is responsible for the deprotonation of L-glutamate. Furthermore, the V[sub max]/K[sub m] isotope effect for the C73S mutant in the D —> L reaction direction is larger than that observed for wild type enzyme and smaller in the L —> D reaction direction. The opposite trend is observed for the C184S mutant. Presumably, an asymmetry in the reaction profile is induced by the mutation making the deprotonation step involving the serine residue more cleanly rate determining. This result supports the assigned roles for each of the cysteine residues of glutamate racemase. Further experiments explore the importance of the four strictly conserved residues, AsplO, Asp36, Glul52 and Hisl86, by preparing appropriate mutant enzymes. The effect of each of the D36N and E152Q mutations is to reduce k[sub cat] by a modest factor of ~2- to 3-fold and suggests these residues are not important to catalysis. They do appear to be involved in binding since there is an increase in the K[sub m] value for each of these mutants. The k[sub cat] values for the D10N and H186N mutants, however, are decreased by three orders of magnitude relative to wild type enzyme implying an important catalytic role for these residues. The V[sub max] isotope effects for D10N and H186N are affected in each reaction direction with the ratio between the isotope effects increased to 2.20 ± 0.18 for D10N relative to the wild type ratio of 1.40 ± 0.34, and decreased to 0.70 ± 0.05 for H186N. A possible role for AsplO and Hisl86 is to stabilize the thiolate form of Cys73 and Cysl84, respectively.Science, Faculty ofChemistry, Department ofGraduat

Analysis and critical review of ICH Q8, Q9 and Q10 from a generic pharmaceutical industry view point

Generic industry aims to produce safe, efficient, built-in quality medicines that will satisfy patients’ requirements and will be competitive on the market. In this paper, assessment of the need for quality by design (QbD) and process analytical technology (PAT) implementation by generic industry was made, by analysis of the ICH Q8, Q9 and Q10 guidelines and their implementation in European regulation. The review of the guidelines indicates differences in the life cycle of a generic medicine, leading to a final conclusion in terms of generic industry. PAT provides statistical analysis and real time quality monitoring, as the basis for proactive quality management. Using QbD/PAT, quality is proved and improved throughout the entire life cycle. Better understanding of the product and processes within a defined design space leads to easier proof of built-in quality throughout the life cycle of the medicine, faster and easier regulatory evaluation, faster time to market, as well as post marketing savings regarding costs and time. Implementation of QbD/PAT as a systematic approach together with risk assessment as part of quality management system is a useful challenge to the generic industry and gives an opportunity for technological, temporal, financial and quality improvement. It was concluded that having in mind its’ own manufacturing capabilities the applicant should optimize the implementation of QbD in accordance with current good manufacturing practice guidelines. Implementation of QbD/PAT is an innovative challenge for the generic industry. Managing pharmaceutical quality system allows the top management to make right decisions at the right time

Medical device risk management and its economic impact

The importance of medical devices in everyday users/patients lives is imensse. This is the reason why emphasis must be put on safety during their use. Satisfactory safety level can be achived by implementation of quality and risk management standards. Medical device manufacturers must learn to deal with the potential risks by using theoretical and practical examples and measures in order to protect their users/patients and themselves from suffering huge losses arising from adverse events or recall of their products. The best moment for implementation of risk management methods and analysis begins from the device design and development through manufacturing, sales and distribution. These way medical device manufacturers will succseed in protecting their users/patients from serious adverse events and at the same time protect their brand and society status, while minimizing economic losses