Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1–471 and 509–616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441–471) in the crystal structurewraps around an /-barrel formed by residues 1–440. Thesecond (residues 472–508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466–512 (GA1), 485–512 (GA2) and 466–483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities.     

Hybrid valve structure for high-throughput, low-volume liquid-handling applications

A hybrid valve that integrates precision microfluidics for fluid routing, high-speed valving for fluid switching, and reagent-jetting devices for metering the fluid dispenses is described. The hybrid valve enables parallel switching between aspiration and dispense modes for multiple sample streams. This unique valve structure addresses many of the concerns with handling microscale volumes, including efficient use of samples, degradation of ink jet valves and speed of operation. A broad range of volumes can be manipulated with excellent reproducibility. The hybrid valve can be configured for a variety of applications. Pick-and-place aspiration and dispensing of unique reagents and rapid dispensing of a common reagent are possible. Together these features lead to higher-speed transfer of smaller volumes of reagent.     

Excretion of volatile ¹³¹I from rats following administration of Na¹³¹I or MIBG-¹³¹I

Current practice for radiation protection associated with (131)I therapy mainly focuses on external and internal exposure caused by physical contamination of the hospital staff, other patients and family members. However, if volatile (131)I is excreted by the treated patients, these individuals could also be exposed through inhalation of (131)I. This study quantifies the amount of volatile (131)I excreted by rats after intravenous administration of metaiodobenzylguanidine (MIBG)-(131)I or Na(131)I, the two most common forms of (131)I therapy. The results indicate that in 4 d following administration, the total excretion of volatile (131)I was 0.036 and 0.17 % of the administered activities of MIBG-(131)I and Na(131)I, respectively. As administered activities for (131)I therapy are typically of the order of 1-10 GBq, the overall excretion of volatile (131)I from a patient can be as high as 20 MBq. As a result, a family member can receive up to 0.07 mSv committed effective dose from inhaling the volatile (131)I excreted by the patient.     

Considerations and preliminary design of patient exposure registry

To aid in protecting patients from unnecessary exposures and to reduce radiation burdens to the public, a system for tracking a patient's medical exposure history and related radiation doses would be a useful tool. A patient-centred exposure registry, the Patient Exposure Registry (PER), is a mechanism that provides this tracking. This article outlines the objectives of the proposed Canadian PER together with considerations and preliminary design of the registry. Implementation strategy is discussed. The strategy will allow many initiatives progressing in parallel such as backward data mining and forward development in order to make this important registry a reality in the near future.     

Near-infrared spectroscopy for cocrystal screening. A comparative study with Raman spectroscopy

Near-infrared (NIR) spectroscopy is a well-established technique for solid-state analysis, providing fast, noninvasive measurements. The use of NIR spectroscopy for polymorph screening and the associated advantages have recently been demonstrated. The objective of this work was to evaluate the analytical potential of NIR spectroscopy for cocrystal screening using Raman spectroscopy as a comparative method. Indomethacin was used as the parent molecule, while saccharin and l-aspartic acid were chosen as guest molecules. Molar ratios of 1:1 for each system were subjected to two types of preparative methods. In the case of saccharin, liquid-assisted cogrinding as well as cocrystallization from solution resulted in a stable 1:1 cocrystalline phase termed IND-SAC cocrystal. For l-aspartic acid, the solution-based method resulted in a polymorphic transition of indomethacin into the metastable alpha form retained in a physical mixture with the guest molecule, while liquid-assisted cogrinding did not induce any changes in the crystal lattice. The good chemical peak selectivity of Raman spectroscopy allowed a straightforward interpretation of sample data by analyzing peak positions and comparing to those of pure references. In addition, Raman spectroscopy provided additional information on the crystal structure of the IND-SAC cocrystal. The broad spectral line shapes of NIR spectra make visual interpretation of the spectra difficult, and consequently, multivariate modeling by principal component analysis (PCA) was applied. Successful use of NIR/PCA was possible only through the inclusion of a set of reference mixtures of parent and guest molecules representing possible solid-state outcomes from the cocrystal screening. The practical hurdle related to the need for reference mixtures seems to restrict the applicability of NIR spectroscopy in cocrystal screening.     

Starch-binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms,GAI [from the N-terminus, catalytic domain + linker + starch-bindingdomain (SBD)] and GAII (catalytic domain + linker). These domainswere shuffled to make RGAI (SBD + linker + catalytic domain),RGAIL (SBD + catalytic domain) and RGAII (linker + catalyticdomain), with domains defined by function rather than by tertiarystructure. In addition, Paenibacillus macerans cyclomaltodextringlucanotransferase SBD replaced the closely related A.nigerGA SBD to give GAE. Soluble starch hydrolysis rates decreasedas RGAII GAII GAI > RGAIL RGAI GAE. Insoluble starchhydrolysis rates were GAI > RGAIL > RGAI >> GAE RGAII > GAII, while insoluble starch-binding capacitieswere GAI > RGAI > RGAIL > RGAII > GAII > GAE.These results indicate that: (i) moving the SBD to the N-terminusor replacing the native SBD somewhat affects soluble starchhydrolysis; (ii) SBD location significantly affects insolublestarch binding and hydrolysis; (iii) insoluble starch hydrolysisis imperfectly correlated with its binding by the SBD; and (iv)placing the P.macerans cyclomaltodextrin glucanotransferaseSBD at the end of a linker, instead of closely associated withthe rest of the enzyme, severely reduces its ability to bindand hydrolyze insoluble starch. Received June 20, 2002; revised May 23, 2003; accepted June 6, 2003.     

RNA‐Cleaving Deoxyribozyme Sensor for Nucleic Acid Analysis: The Limit of Detection

Along with biocompatibility, chemical stability, and simplicity of structural prediction and modification, deoxyribozyme‐based molecular sensors have the potential of an improved detection limit due to their ability to catalytically amplify signal. This study contributes to the understanding of the factors responsible for the limit of detection (LOD) of RNA‐cleaving deoxyribozyme sensors. A new sensor that detects specific DNA/RNA sequences was designed from deoxyribozyme OA‐II [Chiuman, W.; Li, Y. (2006) J. Mol. Biol. 357, 748–754]. The sensor architecture allows for a unique combination of high selectivity, low LOD and the convenience of fluorescent signal monitoring in homogeneous solution. The LOD of the sensor was found to be ～1.6×10^?10 M after 3 h of incubation. An equation that allows estimation of the lowest theoretical LOD using characteristics of parent deoxyribozymes and their fluorogenic substrates was derived and experimentally verified. According to the equation, “catalytically perfect” enzymes can serve as scaffolds for the design of sensors with the LOD not lower than ～2×10^?15 M after 3 h of incubation. A new value termed the detection efficiency (DE) is suggested as a time‐independent characteristic of a sensor's sensitivity. The expressions for the theoretical LOD and DE can be used to evaluate nucleic acid and protein enzymes for their application as biosensing platforms.