Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis

We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.     

Changes in cytochrome P450 molecular species in rat liver in chloroform intoxication

The effect of CHCl3 on the composition of hepatic microsomal cytochrome P450 species was compared with that of CCl4 in rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC). The administration of CHCl3 hardly affected cytochrome P450 content in non-treated rat liver, but caused a similar degree of depletion in the content as observed after CCl4 administration in PB-pretreated rats. In the pretreatment with 3MC, the administration of CHCl3 brought about a marked decrease in the content to 24% of control after 12 hr, while CCl4 reduced the content only to one-half of control. It was demonstrated by SDS-polyacrylamide gel electrophoresis and Whatman DE-52 anion-exchange chromatography that 3MC-induced P450 species decreased with CHCl3, while it was affected little by CCl4 treatment. The activity of benzo[a]pyrene hydroxylase was altered together with the change in the content of cytochrome P450 species. The administration of CHCl3 to PB-pretreated rats caused the depletion in PB-induced P450. These findings indicate that cytochrome P450 species induced with 3MC as well as PB are highly susceptible to CHCl3 intoxication, whereas the administration of CCl4 depletes the PB-induced species without affecting the 3MC-induced species.     

Initial perfusate purification during subnormothermic machine perfusion for porcine liver donated after cardiac death

Journal of Artificial Organs - Improvement of machine perfusion (MP) technologies is required to enhance organ quality for donor after cardiac death (DCD) grafts. Installing a dialyzer or a filter...     

Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human beta-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10-15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.