Evaluation of Polycation-Stabilized Lactate Oxidase in a Silica Sol–Gel as a Biosensor Platform

 Whereas glucose oxidase and related proteins are encapsulated readily in silica sol–gels, α-hydroxy enzymes such as lactate oxidase (LOx), are reported to be damaged by electrostatic interaction with these matrices. Based on a previous report, poly(ethyleneimine), PEI, was evaluated as a protecting compound under conditions suited to analytical measurements. With LOx and PEI co-encapsulated in a silica sol–gel, the enzyme retained 62% of its initial activity after 20 days. In the absence of PEI, activity was lost during the processing. Batch analytical measurements with enzyme-doped sol–gel yielded a linear response over the range 0.5–2.0 mM lactate and a detection limit of 0.03 mM lactate. Both simple incorporation of LOx in a silica sol–gel and an alternative protection method, blocking the ion-exchange sites on silica with La(III), failed. These negative results supported the hypothesis that the efficacy of PEI was related to its formation of a protective sheath around the enzyme. Author for correspondence. E-mail: coxja@muohio.edu Received July 29, 2002; accepted December 15, 2002 Published online May 19, 2003     

The influence of synthetic conditions on the stability of methotrexate-monoclonal antibody conjugates determined by reversed phase high performance liquid chromatography.

Methotrexate (MTX) has been convalently attached to an IgG-type monoclonal antibody (791T/36) directed to tumour-associated antigen gp72. Conjugates were synthesized by the active ester method using MTX N-succinimidyl ester at various pH values (7.5-10.5). Following purification by gel filtration, high performance liquid chromatography was used to assess the free drug or its derivatives in samples of MTX-791T/36 conjugates previously treated (or not) with hydroxylamine. Quantitative analysis, performed on a reversed phase column (pore size 300 A) with isocratic acetonitrile-sodium acetate buffer (pH 4.8) as mobile phase, indicated no detectable amount of free methotrexate in hydroxylamine-treated conjugates even six months after their preparation. Similar observations were made with conjugates, whose synthesis were performed at pH greater than or equal to 10. In contrast, the presence of increasing amounts of drug/metabolite could be demonstrated in samples produced at lower pH values. Based on these findings, the pH-dependent kinetics of MTX release has been determined and used to design conditions under which stable MTX-791T/36 conjugates could be prepared without hydroxylamine reaction.     

Learning to Make Sense of Division of Fractions: One K–8 Preservice Teacher's Perspective

Current reform documents in mathematics education recommend that teachers help students develop both conceptual and procedural understandings. However, teachers often do not possess the in-depth mathematical reasoning necessary to accomplish this goal. The purpose of this article is to provide one way in which preservice teachers can come to better understand the mathematics they will teach.     

Innately transitive subgroups of wreath products in product action

A permutation group is innately transitive if it has a transitive minimal normal subgroup, which is referred to as a plinth. We study the class of finite, innately transitive permutation groups that can be embedded into wreath products in product action. This investigation is carried out by observing that such a wreath product preserves a natural Cartesian decomposition of the underlying set. Previously we classified the possible embeddings in the case where the plinth is simple. Here we extend that classification and identify several different types of Cartesian decompositions that can be preserved by an innately transitive group with a non-abelian plinth. These different types of decompositions lead to different types of embeddings of the acting group into wreath products in product action. We also obtain a full characterisation of embeddings of innately transitive groups with diagonal type into such wreath products.

     

New Chiral Kemp's Acid Diamides for Chiral Amine Recognition by 1 H NMR

Two Kemp's acid diamides were synthesized and applied to chiral amine recognition using ¹H NMR analysis. One derivative based on 1-(1-naphthyl)ethylamine had good chiral recognition of six amines and was useful to determine the optical purity for three amines, i.e., methylbenzylamine, 1-(1-naphthyl)ethylamine and 1- henylpropylamine,however, the cyclohexylethylamine derivative showed little discrimination for the amines studied. Together with the results for alkylamines, it was shown that aromatic structure was important for aromatic shielding anisotropy and –– interactions between host and guest. The structure of the 1-(1-naphthyl)ethylamine derivative in solution was also considered based on ¹H NMR data and computer simulation.     

Determination of organic carbon in water with a silver-catalyzed peroxydisulfate wet chemical oxidation method

The application of Ag(I) catalysis of S₂O₈^2? to the oxidation of organic carbon to CO₂ for determination of dissolved organic carbon in aqueous samples is described. The resulting method combines to a significant degree the speed of high-temperature combustion methods and the sensitivity of wet chemical oxidation. For some samples, a higher oxidation efficiency has been observed than with an uncatalyzed wet chemical oxidation method.     

Synthesis of polymer-bound hemoglobin samples

Dextran and hydroxyethyl starch have been chemically modified to give aldehyde-substituted polymers. These modified polymers were synthesised either by periodate oxidation of the starting polymer or by attachment of gluteraldehyde to amino-substituted side chains on the polymer. When such modified starches were allowed to react with hemoglobin soluble polymer-bound hemoglobins were formed. These were shown to be capable of binding oxygen but the observed oxygen-binding curves were shifted to the left relative to unbound hemoglobin. Heart perfusion experiments indicate that these polymer-bound hemoglobins are not suitable for use as blood substitutes     

The Raman active internal vibrational modes of potassium nitrate

The Raman active internal vibrational modes of single crystal orthorhombic potassium nitrate have been studied in various polarizations. The full multiplet structure predicted by factor group analysis for the v₂ and v₃ regions has been observed for the first time. The expected site group splitting of the v₄ mode was not observed and can be assumed to be less than 0.5 cm^?1.     

Anion-controlled nuclearity in nickel complexes with potentially dinucleating, poly(oxime) amine ligands

Two new ligands consisting of bis(oxime) amine units tethered by a bridge have been synthesized. Their nickel chloride and nickel nitrate complexes have also been synthesized and characterized by X-ray crystallography, FTIR, mass spectrometry, and elemental analysis. One of these ligands, L1 (N,N,N',N'-tetra(1-propan-2-onyl oxime)-diamino-m-xylene), is always dinucleating, while the other ligand, L2 (N,N,N',N'-tetra(1-propan-2-onyl-oxime)-1,3-diaminopropane), shows an unusual anion dependence on the nuclearity. When nickel chloride is used, the ligand acts in a dinucleating manner and coordinates two nickels; however, when nickel nitrate is used, the ligand acts in a monodentate fashion and coordinates only one nickel. Once the mononuclear complex is formed, it is not possible to add a second nickel if Ni(NO(3))(2) is used as the nickel source; it is possible, however, to add a second nickel if NiCl(2) is used as the nickel source. The dinuclear complex can be converted to the mononuclear one by either using silver nitrate to exchange the chloride anions for nitrates or by dissolving the complex in water. Ni(2)(L1)Cl(4)(DMF)(2).DMF: orthorhombic, P2(1)2(1)2(1), a = 12.2524(11) A, b = 16.6145(15) A, c = 20.1234(19) A, V = 4096.5(6) A(3), Z = 4. [Ni(2)(L2)Cl(4)(DMF)](2).2DMF: triclinic, P-1, a = 12.5347(5) A, b = 12.5403(5) A, c = 14.3504(6) A, alpha = 67.348(1) degrees , beta = 69.705(1) degrees , gamma = 81.549(1) degrees , V = 1952.25(14) A(3), Z = 1. Ni(L2).(NO(3))(2): monoclinic, P2(1)/n, a = 9.6738(3) A, b = 30.2229(9) A, c = 15.8238(5) A, beta = 97.995(1) degrees , V = 4581.4(2) A(3), Z = 8.     

NMR and X-ray crystallography, complementary tools in structural proteomics of small proteins

NMR spectroscopy and X-ray crystallography, the two primary experimental methods for protein structure determination at high resolution, have different advantages and disadvantages in terms of sample preparation and data collection and analysis. It is therefore of interest to assess their complementarity when applied to small proteins. Structural genomics/proteomics projects provide an ideal opportunity to make such comparisons as they generate data in a systematic manner for large enough numbers of proteins to allow firm conclusions to be drawn. Here we report a comparison for 263 unique proteins screened by both NMR spectroscopy and X-ray crystallography in our structural proteomics pipeline. Only 21 targets (8%) were deemed amenable to both methods based on an initial 2D 15N-HSQC NMR spectrum and optimized crystallization trials. However, the use of both methods in the pipeline increased the total number of targets amenable to structure determination to 107, with 43 amenable to NMR only and 43 amenable to X-ray crystallographic methods only. We did not observe a correlation between 15N-HSQC spectral quality and the success of the same protein in crystallization screens. Similar results were found for an independent set of 159 proteins as reported in the accompanying paper by Snyder et al. Thus, we conclude that both methods are highly complementary, and in order to increase the number of proteins suited for structure determination, we suggest that both methods be used in parallel in screening of all small proteins for structure determination.