Screening for soluble methane monooxygenase in methanotrophic bacteria using combined molecular and biochemical methods for hydroxylase

detection.
Grosse S, Mueller C, Rogge G, Wendlandt KD, Miguez CB, Kleber HP.

Source
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada. Stephan.Grosse@nrc.ca

Abstract
Three well known methanotrophic bacteria (Methylosinus trichosporium OB3b, Methylocystis sp. WI 14, and Methylocystis sp. GB 25) and three newly isolated methanotrophic bacteria (Methylocystis sp. WI 11, Methylocystis sp. X, and FI-9) were screened for sMMO considering the existence of hydroxylase (component A) genes as well as its gene expression. For these purposes monoclonal antibodies that specifically recognize each subunit of the hydroxylase of Methylocystis sp. WI 14 (alpha-subunit [9E5/F2], beta-subunit [4E2/G11], gamma-subunit [10G3/D7]) were produced. PCR amplification using well known primers showed that the hydroxylase encoding genes appear to be only present in M. trichosporium OB3b, Methylocystis sp. WI 11 and WI 14, and in the isolate FI-9. Western and ELISA analysis using the monoclonal antibodies revealed that all subunits of hydroxylase were present. However, in FI-9, only the alpha-subunit of the hydroxylase might be expressed. Surprisingly, in Methylocystis sp. GB 25, where no sMMO activity and no amplification with sMMO specific primers was obtained, the antibody 4E2/G11 recognized a protein band with exactly the same molecular mass as the beta-subunit of the hydroxylase. Methylocystis sp. X showed no positive reaction in any of the tests. In combination with the detection methods currently used, the described antibodies provide a powerful tool for detecting even partially expressed hydroxylase genes.

Preparation and Physicochemical Characterization of Acyclovir Cocrystals with Improved Dissolution Properties.
Bruni G, Maietta M, Maggi L, Mustarelli P, Ferrara C, Berbenni V, Milanese C, Girella A, Marini A.

Source
C.S.G.I., Department of Chemistry, Physical-Chemistry Section, University of Pavia, Pavia, Italy.

Abstract
Acyclovir is a well-known antiviral agent. It can be administered in very high doses (from 200 to 1000 mg even three-four times daily). It has absorption problems mainly due to its poor solubility in water (about 0.2 g/100 mL at 25C) and its oral bioavailability is approximately 15%-20% with a half-life of about 3 h. To improve acyclovir solubility and/or its dissolution properties, two cocrystals of this drug were successfully produced with glutaric acid (AGA1:1) and fumaric acid (AFA1:1) as conformers, using a cogrinding method. Their effective formation was investigated by a broad range of techniques: thermal analysis, Fourier transform infrared spectroscopy, X-ray powder diffraction, solid state nuclear magnetic resonance, and scanning electron microscopy coupled with energy dispersive X-ray spectrometry. The water solubility of the AGA1:1 cocrystal was not improved in comparison to acyclovir, while AFA1:1 showed a slight increased solubility at equilibrium. The main difference was detected in terms of intrinsic dissolution rates (IDR). The IDR of the new phases were much faster compared with acyclovir, particularly at neutral pH. AFA1:1 showed the most rapid dissolution behavior in water; within 10 min, the drug was released completely, while just 60% of acyclovir was dissolved in 1 h. 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci. 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

KEYWORDS:
Acyclovir, SEM-EDS, calorimetry (DSC), cocrystal, dissolution rate, fumaric acid, glutaric acid, grinding, solid state NMR, solubility

Interaction of antidepressant drug, clomipramine, with model and biological stratum corneum membrane as studied by electron paramagnetic resonance.
Yonar D, Horasan N, Pakta DD, Abramovi Z, Strancar J, Snnetiolu MM, Sentjurc M.

Source
Hacettepe University, Department of Physics Engineering, Ankara, 06800, Turkey.

Abstract
The interactions of tricyclic antidepressant drug, clomipramine (CLO), with pig ear stratum corneum (SC) and model membranes were investigated by electron paramagnetic resonance (EPR) spin labeling to get some insight into the possible application of this drug in transdermal delivery. The changes in membrane characteristics caused by CLO in the regions that are close to the water-lipid interfaces and the central parts of the membranes were searched. The experimental results were supported by computer simulation of EPR spectra, which showed heterogeneity of the membranes composed of regions with different fluidity characteristics. CLO was effective in both parts of the layers, indicating intercalation of the drug into model membranes as well as into the pig ear SC. Introduction of various molar ratios of CLO caused a decrease in the order parameter and an increase in the rotational diffusion of nitroxide moiety in different membrane regions as well as an increase in the polarity of spin probe environment. It also changed the number of resolved spectral components, which reflects the heterogeneity of the membrane. The fluidizing effect of CLO on pig ear SC throughout the whole membrane layers indicates that CLO penetrates into the SC, which is important for its transdermal delivery. 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3762-3772, 2013. 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

KEYWORDS:
EPR spin labeling, antidepressant, diffusion, liposomes, simulation, spectroscopy, stratum corneum, transdermal drug delivery