Evaluation of the accuracy of urine analyzers in dogs and cats.

The accuracy of urine analyzers used for dogs and cats has remained uncertain. This study examines the agreement between results of urine analysis obtained using two devices marketed for animals and for humans and the results of quantitative biochemical analysis. The degrees of concordance for bilirubin and ketones in the same category were ~80%, but for pH these were only ~60% in dogs and cats. Degrees of concordance for protein and the UP/C ratio clearly differed between the devices for animals and humans. We found that values for bilirubin and ketones obtained using urine analyzers may be reliable, but pH is unlikely to be accurate enough to be clinically useful for dogs and cats.

Potential use of lactosylated dendrimer (G3)/α-cyclodextrin conjugates as hepatocyte-specific siRNA carriers for the treatment of familial amyloidotic polyneuropathy.

To reveal the potential use of lactosylated-dendrimer (G3) conjugates with α-cyclodextrin (Lac-α-CDE (G3)) as novel hepatocyte-specific siRNA carriers in order to treat transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP), we evaluated the RNAi effect of siRNA complexes with Lac-α-CDE (G3) both in vitro and in vivo. Herein, we targeted TTR gene expression because TTR-related FAP was often caused by amyloidogenic TTR (ATTR), which mainly expresses in hepatocytes. Lac-α-CDE (G3, average degree of substitution of lactose (DSL) 1.2)/siRNA complex had a potent RNAi effect against TTR gene expression through adequate physicochemical properties, asialoglycoprotein receptor (ASGP-R)-mediated cellular uptake, efficient endosomal escape and the delivery of the siRNA complex to cytoplasm, but not nucleus, with negligible cytotoxicity. Lac-α-CDE (G3, DSL 1.2)/siRNA complex had the potential to induce the in vivo RNAi effect after intravenous administration in the liver of mice. The blood chemistry values in the α-CDE (G3) and Lac-α-CDE (G3, DSL 1.2) systems were almost equivalent to those in the control system (5% mannitol solution). Taken together, these results suggest that Lac-α-CDE (G3, DSL 1.2) has the potential for a novel hepatocyte-selective siRNA carrier in vitro and in vivo, and has a possibility as a therapeutic tool for FAP to the liver transplantation.

Antimicrobial susceptibility of Escherichia coli isolated from feces of wild cranes migrating to Kagoshima, Japan.

Susceptibility to 13 antimicrobial agents was examined for 138 Escherichia coli isolates obtained from 192 fecal samples of wild cranes that migrated for wintering to the Izumi plain, Kagoshima prefecture in Japan. The numbers of isolates that were resistant to the antimicrobials used in this study are as follows: oxytetracycline (OTC), 22 isolates; minocycline, 7 isolates; ampicillin (ABPC), 4 isolates; nalidixic acid, 4 isolates; enrofloxacin, 2 isolates; kanamycin, one isolate. Multidrug resistant isolates exhibiting 2-4 drug resistances were obtained. All of the OTC-resistant isolates carried either the tet (A) or tet(B) gene. The bla(TEM) gene was found in all of the ABPC-resistant isolates.

In vitro and in vivo gene delivery mediated by Lactosylated dendrimer/alpha-cyclodextrin conjugates (G2) into hepatocytes.

The purpose of this study is to evaluate in vitro and in vivo gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugates with alpha-cyclodextrin (alpha-CDE (G2)) bearing lactose (Lac-alpha-CDE) with various degrees of substitution of the lactose moiety (DSL) as a novel hepatocyte-selective carrier in hepatocytes. Lac-alpha-CDE (DSL 2.6) was found to have much higher gene transfer activity than dendrimer, alpha-CDE, Lac-alpha-CDE (DSL 1.2, 4.6, 6.2 and 10.2) and lactosylated dendrimer (Lac-dendrimer, DSL 2.4) in HepG2 cells, which are dependent on the expression of cell-surface asialoglycoprotein receptor (ASGP-R), reflecting the cellular association of the plasmid DNA (pDNA) complexes. The physicochemical properties of pDNA complex with Lac-alpha-CDE (DSL 2.6) were almost comparable to that with alpha-CDE. Lac-alpha-CDE (DSL 2.6) provided negligible cytotoxicity up to a charge ratio of 150 in HepG2 cells. Lac-alpha-CDE (DSL 2.6) provided gene transfer activity higher than jetPEI-Hepatocyte to hepatocytes with much less changes of blood chemistry values 12h after intravenous administration in mice. These results suggest the potential use of Lac-alpha-CDE (DSL 2.6) as a non-viral vector for gene delivery toward hepatocytes.

Slow-release system of pegylated lysozyme utilizing formation of polypseudorotaxanes with cyclodextrins.

Poly(ethylene glycol) (PEG, MW 2200) chains were introduced into lysozyme molecule. The resulting pegylated lysozyme formed polypseudorotaxanes with alpha- and gamma-cyclodextrins (alpha- and gamma-CyDs, respectively), by inserting one PEG chain in the alpha-CyD cavity and two PEG chains in the gamma-CyD cavity. The pegylated lysozyme/CyD polypseudorotaxanes were less soluble in water and the release rate of the pegylated protein decreased in the order of the pegylated lysozyme>the gamma-CyD polypseudorotaxane>the alpha-CyD polypseudorotaxane. The enzymatic activity of the pegylated lysozyme released from the polypseudorotaxanes was the same as that of the pegylated protein alone, indicating no decrease in the activity through the polypseudorotaxane formation. The results indicate that the pegylated lysozyme/CyD polypseudorotaxanes can work as a slow-release system, and the polypseudorotaxane formation with CyDs may serve as a new strategy for the preparation of slow-release system of pegylated proteins and peptides.

Enhancement of gene transfer activity mediated by mannosylated dendrimer/alpha-cyclodextrin conjugate (generation 3, G3).

To enhance gene transfer activity of dendrimers, we prepared its conjugate (generation 3, G3) with alpha-cyclodextrin bearing mannose (Man-alpha-CDE conjugates) with various degrees of substitution of the mannose moiety (DSM5, 10, 13, 20) and compared their cytotoxicity and gene transfer activity, and elucidated the enhancing mechanism for the activity. Of the various carriers used here, Man-alpha-CDE conjugate (G3, DSM10) provided the highest gene transfer activity in NR8383, A549, NIH3T3 and HepG2 cells, being independent of the expression of mannose receptors. Gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) was not decreased by the addition of 10% serum in A549 cells. Cytotoxicity of the polyplex with Man-alpha-CDE conjugates (G3, DSM10) was not observed in A549 and NIH3T3 cells up to the charge ratio of 200/1 (carrier/pDNA). The gel mobility and particle size of polyplex with Man-alpha-CDE conjugate (G3, DSM10) were relevant to those with alpha-CDE conjugate (G3), but zeta-potential, DNase I stability, pDNA condensation of the former polyplex were somewhat different from those of the latter one. Cellular association of polyplex with Man-alpha-CDE conjugate (G3, DSM10) was almost comparable to that with dendrimer (G3) complex and alpha-CDE conjugate (G3). The addition of mannan and mannose attenuated gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) in A549 cells. Alexa-pDNA complex with TRITC-Man-alpha-CDE conjugate (G3, DSM10), but not the complex with TRITC-alpha-CDE conjugate (G3), was found to translocate to nucleus at 24 h after incubation in A549 cells. HVJ-E vector including mannan, but neither the vector alone nor the vector including dextran, suppressed the nuclear localization of TRITC-Man-alpha-CDE conjugate (G3, DSM10) to a striking degree after 24 h incubation in A549 cells. These results suggest that Man-alpha-CDE conjugate (G3, DSM10) has less cytotoxicity and prominent gene transfer activity through not only its serum resistant and endosome-escaping abilities but also nuclear localization ability.