Detection of tulobuterol crystal in transdermal patches using terahertz pulsed spectroscopy and imaging.

Applicability of a Terahertz Pulsed Spectroscopy (TPS) and a Terahertz Pulsed Imaging (TPI) for detection of tulobuterol (TBR) crystals in transdermal patches was investigated. Because TBR has high permeability in dermis, crystalline TBR in patch matrices contributes to controlling the release rate of TBR from a matrix. Therefore, crystalline TBR is one of the important factors for quality control of TBR transdermal tapes. A model tape that includes 5 w/w%, 10 w/w%, 20 w/w% or 30 w/w% of TBR was measured by TPS/TPI. TBR crystals in the matrices were successfully detected by TPI. Identification of TBR in an image of a crystal-like mass was done by comparison between the spectra of tapes and a TBR standard substance. These results indicate that TPS and TPI are applicable to identifying crystalline lumps of an active drug in tapes for quality control.

Chemical mapping of tulobuterol in transdermal tapes using microscopic laser Raman spectroscopy.

Microscopic Laser Raman Spectroscopy and Mapping (MLRSM) technique was used to investigate the distribution of tulobuterol (TBR) crystals in transdermal tapes. TBR is one of suitable compounds for the transdermal pharmaceuticals because it has high permeability into skin. In case of TBR transdermal tapes, some commercial products also contain TBR crystals in order to control a release rate from a matrix. Therefore, the presence of TBR crystals in the matrix is a critical factor for quality assurance of this type of TDDS tapes. The model tapes prepared here employed two kinds of matrices, i.e., rubber or acrylic, which are generally used for transdermal pharmaceuticals. TBR crystals in the matrix were observed by MLRSM. Accurate observation of the distribution of TBR in the tapes was achieved by creating a Raman chemical map based on detecting unique TBR peak in each pixel. Moreover, differences in the growth of TBR crystals in the two kinds of matrices were detected by microscopic observation. MLRSM also enabled the detection of TBR crystals in commercial products. The present findings suggest that Raman micro-spectroscopic analysis would be very useful for verifying and/or assessing the quality of transdermal pharmaceuticals in development, as well as for manufacturing process control.

Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process.

AIMS: The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification. METHODS AND RESULTS: cDNA reverse-transcribed from 16S rRNA was amplified with fluorescent labelled primer for terminal restriction fragment length polymorphism (T-RFLP) analysis and an unlabelled primer for cloning analysis. The terminal restriction fragments (T-RFs) that increased in association with the start of denitrification were determined. These T-RFs were identified by in silico analysis of 16S rRNA sequences obtained from cloning. As a result, it was clearly observed that the bacteria belonging to the genera Hydrogenophaga and Acidovorax increased in number after the start of denitrification. CONCLUSIONS: It was demonstrated that T-RFLP analysis targeting 16S rRNA is appropriate for the daily monitoring of a bacterial community to control wastewater treatment processes. Combination of the results of T-RFLP analysis and 16S rRNA clone library indicated that the bacteria belonging to the genera Hydrogenophaga and Acidovorax play an important role in denitrification. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide new insight to the 16S rRNA level of active denitrifying bacteria in wastewater treatment processes.

Dependence of low-frequency sonophoresis on ultrasound parameters; distance of the horn and intensity.

Sonophoresis at a frequency of 20 kHz has been shown to enhance transdermal drug delivery, a phenomenon referred to as low-frequency sonophoresis. This study provides an investigation of the dependence of low-frequency sonophoresis on various ultrasound parameters, including the distance of the horn from the skin, intensity, and frequency. We performed in vitro experiments with full thickness pig skin to measure enhancements of skin conductivity and drug permeability. Ultrasound was applied to pretreat the skin using a sonicator operating at a frequency of either 20 or 40 kHz. We also measured pitting of aluminum foil to measure cavitation, which is the principal mechanism of low-frequency sonophoresis. The skin conductivity enhancement was found to be inversely proportional to the distance of the horn from the skin. As the intensity increased, skin conductivity enhancement also increased up to a certain threshold, and then dropped off. The intensities (I(max)) at which maximum enhancement occur are about 14 W/cm2 for 20 kHz and 17 W/cm2 for 40 kHz. These findings may be useful in optimizing low-frequency sonophoresis. Overall, the dependence of transport on ultrasound parameters is similar to that of aluminum foil pitting. These results support the role of cavitation in low-frequency sonophoresis.

Determination of threshold energy dose for ultrasound-induced transdermal drug transport.

Low-frequency (20 kHz) ultrasound has been shown to enhance transdermal transport of drugs, a phenomenon referred to as sonophoresis. In this paper, we report the threshold energy dose for ultrasound-induced transdermal drug transport. The threshold was determined by in vitro measurements of the dependence of sonophoretic enhancement on ultrasound parameters, including intensity, duty cycle, and exposure time. While the enhancement varies linearly with ultrasound intensity and exposure times, it is independent of the duty cycle in the range of parameters studied. The enhancement is also directly proportional to the ultrasound energy density once the threshold value is crossed. For full thickness pig skin, the threshold value is about 222 J/cm(2). The overall dependence of transport enhancement on ultrasound parameters is similar to that of cavitation measured in a model system, pitting of aluminum foil. Specifically, the extent of pitting is proportional to ultrasound intensity and exposure time and is independent of duty cycle. Furthermore, the extent of pitting is also proportional to the ultrasound energy density. The similarity between the parametric dependence of transport enhancement and cavitation is consistent with previous findings that cavitation plays the dominant role in sonophoresis.

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus.

We examined the timing and mechanisms of mesodermal and neural determination in Cynops, using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops.

Direct 99mTc labeling of human immunoglobulin with an insoluble macromolecular Sn(II) complex.

An insoluble macromolecular Sn(II)(R-Sn) complex which strongly binds Sn(II) by chelation was applied to the direct 99mTc labeling of human immunoglobulin(IgG) to minimize the influence of Sn(II). 99mTc labeling was achieved at greater than 90% yield simply by the short-term mixing of IgGa containing > 2-SH groups per IgG molecule, 99mTc pertechnetate, and the R-Sn complex in pH 7 solution. The 99mTc-IgGa obtained by this labeling method has high stability based on the thiol-specific binding of 99mTc without transchelation from another weakly bound 99mTc-complex.

Characterization of insoluble macromolecular Sn(II) complex and its application to the 99mTc labeling of human serum albumin-bearing mercapto groups.

A chelating ion exchange resin containing aminophosphonic acid groups was used as a polymer matrix for the preparation of an insoluble macromolecular Sn(II)(R-Sn) complex. Sn(II), which strongly bound to the surface of the polymer matrix by chelation, retained the ability to reduce 99mTc in the R-Sn complex. Human serum albumin-bearing-mercapto groups (HMA) was labeled with 99mTc at pH 2-3 using the R-Sn complex or SnCl2 as reducing agent. The 99mTc-HMA labeled with the R-Sn complex resulted in a higher sustained level of radioactivity in the blood of mice than the 99mTc-HMA labeled with SnCl2. These results suggest that the use of the R-Sn complex minimized Sn(II) contamination of the 99mTc labeling solution and can be used effectively as a reducing agent for 99mTc labeling of proteins containing sulfhydryl groups.

Development of macromolecular Sn(II) complex for preparation of 99Tcm radiopharmaceuticals.

Macromolecular Sn(II) complex (R-Sn) was developed using a chelating resin containing aminophosphonic acid groups as a reducing agent of 99Tcm(VII) in the preparation of 99Tcm radiopharmaceuticals. Since Sn(II) was bonded strongly to the resin by chelation, release of Sn from R-Sn was rarely observed in saline solution. 99Tcm labelling with a yield greater than 90% was performed by mixing R-Sn, 99Tcm pertechnetate solution and a ligand, such as human serum albumin and diethylenetriamine pentaacetic acid, for a short time. The 99Tcm radiopharmaceuticals were almost free from Sn(II) and the reducing activity of R-Sn was quite stable despite long-term storage without any special care.

Differentiation and distribution of annulate lamellae in growing oocytes in the newt, Cynops pyrrhogaster.

Stages of oocyte development in Cynops pyrrogaster are defined, and changes of annulate lamellae in their fine structure, number, sizes and locations during oogenesis are described. The results show that two different types of annulate lamellae occur during oogenesis. One type differentiates in or at the periphery of vesicle-rich cytoplasm at the early stages of vitellogenesis and increases in number and size. The maximum number of about 40 stacks per median section of oocyte is reached at the stage of complete differentiation of the animal and the vegetal hemispheres. In these growing oocytes, all the stacks show elongate appearances and tetragonal arrangements of annuli as common characteristics. A second type of stacks of annulate lamellae is added anew in full-grown oocytes, increasing the number of stacks per median section of the oocyte to about 90. The new stacks occur in close contact with electron-dense bodies in the cytoplasm and have a massive appearance and hexagonal array of annuli. It is suggested that they appear coincidentally with the onset of oocyte maturation. The possible significance of the observed results is discussed.