Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2-1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A (417)) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A (417) was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.     

Quantitative evaluation of lectin‐reactive glycoforms of α1‐acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection

α₁‐Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein‐labeled AGP using lectins with the aid of laser‐induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A‐reactive or Aleuria aurantia lectin‐reactive AGP. Labeled AGP was applied at the anodic end of a fused‐silica capillary (50 μm id, 360 μm od, 27 cm long) coated with linear polyacryloyl‐β‐alanyl‐β‐alanine, and electrophoresis was carried out for about 10 min in 60 mM 3‐morpholinopropane‐1‐sulfonic acid‐NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin‐reactive glycoform peaks from lectin‐non‐reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin‐reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5 mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro‐purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies.     

Experimental assessment of fluorescence microscopy signal enhancement by stimulated emission

The quantity of photons generated during fluorescence microscopy is principally determined by the quantum yield of the fluorescence dyes and the optical power of the excitation beam. However, even though low quantum yields can produce poor images, it is challenging to tune this parameter, while increasing the power of the excitation beam often results in photodamage. Here, we propose the use of stimulated emission (SE) as a means of enhancing both the signal intensity and signal-to-noise ratio during confocal fluorescence microscopy. This work experimentally confirmed that both these factors can be enhanced by SE radiation, through generating a greater number of photons than are associated with the standard fluorescence signal. We also propose the concept of stimulated emission enhancing fluorescence (SEEF) microscopy, which employs both the SE and fluorescence signals, and demonstrate that the intensity of an SEEF signal is greater than those of the individual SE and fluorescence signals.     

A prescribed-energy problem for a conservative singular Hamiltonian system

Communicated by P. Rabinowitz     

Radical-Initiated homopolymerization and copolymerization of methylthiomethyl methacrylate

Methylthiomethyl methacrylate (MtMA) was synthesized and homopolymerized in solution. The poly(MtMA) is readily soluble in benzene, acetone, tetrahydrofuran, and methylene chloride at room temperature. The values of K and a in the Mark–Houwink equation, [η] = KM^a, were found to be K = 2.88 × 10^–5 and a = 0.75 when M = M_w. The glass transition temperature of poly(MtMA) was observed to be 72°C by thermomechanical analysis. Intramolecular anhydride formation occurred when poly(MtMA) was heated to 250–300°C. The kinetics of MtMA homopolymerization was investigated in benzene, using azobisisobutyronitrile as initiator. The rate of polymerization R_p was expressed by R_p = k[AIBN]^0.5[MtMA]^1.05 and the overall activation energy was calculated to be 75.7 kJ/mol. The relative reactivity ratios of MtMA in styrene copolymerizations (r₁ = 0.33, r₂ = 0.55) were obtained. Applying the Q-e scheme led to Q = 1.07 and e = 0.51 for MtMA.     

Preparation of water and alkali durable porous glass membrane coated on porous alumina tubing by sol-gel method

Composite porous glass membranes were prepared by the sol-gel method. A thin porous glass layer, about 2 μm thick, was coated on the surface of the porous ceramic tubing (Al₂O₃:99.9 wt.%, pore diameter: 200 nm). The composition of the porous glass layer of the composite membrane was SiO₂-ZrO₂. Considering from the fact that the desalination ratio of the feed aqueous NaCl solution (NaCl 0.5 wt.%) was about 90% by use of these membranes, they were defect-free. The best composition of the porous glass layer was 70 SiO₂-30 ZrO₂ from the standpoint of preparing membranes. These membranes had a large water and alkali durability. These membranes can be expected to apply to recovering dyes and paints from organic solvents and to be used as a gas separation membrane.     

Enhancing effect of cyclodextrins on nasal absorption of insulin and its duration in rabbits.

The absorption of insulin (from porcine pancreas) in rabbits after the nasal administration of aqueous preparations containing insulin and five kinds of cyclodextrins (CyDs) in phosphate buffer solution at pH 7.0 was investigated. Without CyD, the insulin and glucose levels in plasma were unchanged, whereas a marked increase in the plasma levels of insulin and a decrease in glucose concentrations were observed following the simultaneous administration of insulin and CyD such as alpha- and heptakis (2,6-di-O-methyl)-beta-CyD (DM-beta-CyD). The largest enhancing effect on the nasal absorption of insulin was obtained by DM-beta-CyD. To evaluate the duration of the absorption-enhancing effect of CyDs, preadministration (administration of CyD 0.5, 6, 12 and 24 h before insulin administration) was performed. The area under plasma concentration-time curve (AUC) and Cmax of insulin significantly decreased with the preadministration of DM-beta-CyD 6, 12 and 24 h before nasal administration. The absorption-enhancing effect disappeared 24h after the preadministration. These findings demonstrate that CyDs enhance the nasal absorption of insulin, and the recovery of the membrane transport barrier function in nasal mucosa is achieved, at the latest, 24 h after the administration of CyDs.     

Importance of Debye and Keesom interactions in separating m-xylene and p-xylene in GC-MS analysis utilizing PEG stationary phase

In electron impact gas chromatography (GC)-mass spectrometry analysis of a complex mixture, such as gasoline, two coeluting solutes can be distinguished if each has a unique major ion. The boiling-point difference between m-xylene and p-xylene, which also has identical major ions (mz-1: 91 and 106 Da), is 0.77 degrees C. These cannot be separated even on a crossed-linked polydimethylsiloxane capillary column, which has a minimum of 5000 plates/m. They are separated on a crossed-linked polar polyethylene glycol (PEG) capillary column. GC separation on a stationary phase depends on the relative strengths of solute-solute, stationary phase-stationary phase, and solute-stationary phase interactions. Although the calculated molar electronic polarization and refractivity factor of Lorenz-Lorentz equation for m-xylene and p-xylene are nearly equal because of its greater dipole moment difference (0.30 and 0.02), the calculated orientation polarization of m-xylene is 80 times greater than p-xylene. This implies the dipole reinforcement through inductive polarization by the hydroxyl of PEG stationary phase molecules is greater on m-xylene than p-xylene. In addition, as the permanent dipole moment of m-xylene is 15 times greater than p-xylene, m-xylene has a stronger Keesom interaction with PEG. In order for m-xylene and p-xylene to solvate in PEG, analytes must overcome the PEG-PEG Keesom/hydrogen bonding interaction forces. Physical and chemical parameters indicate that compared with p-xylene, m-xylene has a greater Debye-Keesom interaction tendency with PEG molecules. This is supported by the 0.12-min. retention-time difference between them.     

Multiple periodic solutions of a superlinear forced wave equation

We study the existence of forced vibrations of nonlinear wave equation: (*) $$\begin{array}{*{20}c} {u_{tt} - u_{xx} + g(u) = f(x,t),} & {(x,t) \in (0,\pi ) \times R,} \\ {\begin{array}{*{20}c} {u(0,t) = u(\pi ,t) = 0,} \\ {u(x,t + 2\pi ) = u(x,t),} \\ \end{array} } & {\begin{array}{*{20}c} {t \in R,} \\ {(x,t) \in (0,\pi ) \times R,} \\ \end{array} } \\ \end{array}$$ whereg(ξ)∈C(R,R)is a function with superlinear growth and f(x, t) is a function which is 2π-periodic in t. Under the suitable growth condition on g(ξ), we prove the existence of infinitely many solution of (*) for any given f(x, t).