Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system

A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument. The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (n = 28) of serially diluted Aspergillus fumigatus conidia (10(5) to 0 CFU/ml). Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol. The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sensitivity when DNA was extracted manually; in 9 of 28 runs, we could achieve a higher sensitivity of 1 CFU/ml blood, which was found to be significant (p 相似文献   

Physical Deletion of the p53 Gene in Bladder Cancer: Detection by Fluorescence in Situ Hybridization

To understand better the role of physical p53 deletion in bladder cancer, 106 formalin-fixed and 45 unfixed bladder tumors were examined using fluorescence in situ hybridization. Probes for centromere 17 and the p53 locus were hybridized simultaneously to interphase tumor cells to analyze p53 and chromosome 17 copy number on a cell by cell basis. 17p deletion was found in four of 43 pTa tumors, 18 of 43 pT1 tumors and 29 of 58 pT2-4 tumors (P = 0.0001). 17p deletion was also highly correlated with grade (P = 0.0001) and with p53 immunostaining (P = 0.0005). Chromosome 17 polysomy was associated with stage, grade, 17p deletions, and p53 immunostaining (P = 0.0001). The strong difference in centromere 17 copy number and 17p deletions between pTa and pT1 tumors supports a relevant biological distinction between pTa and pT1 tumors.     

BCG Antibody Profiles in Tuberculoid and Lepromatous Leprosy

In sera from 12 patients with polar tuberculoid leprosy, 12 with subpolar tuberculoid leprosy, and 16 with lepromatous leprosy were demonstrated a total number of 125 anti-BCG precipitins by means of crossed immunoelectrophoresis with intermediate gel. Up to 14 different precipitins were found in individual sera, and the complexity in antibody response was higher than previously realized. The specificity of 69% of the antibodies was defined, and these antibodies were titrated in three arbitrary titer units. A highly significant difference (P < 0.002) was found in antibody response between the tuberculoid and the lepromatous group. Due to simplicity, sensitivity, and high resolution, the method used is a promising tool for providing exact data to be used as guidelines for purification of important individual mycobacterial antigens. The need for reference antisera is emphasized.     

Transgenic expression of a CD46 (membrane cofactor protein) minigene: Studies of xenotransplantation and measles virus infection

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.     

Autoimmune associated recurrent abortions

A possible relationship between recurrent spontaneous abortionsand autoimmune abnormalities was studied. Eight serologicalautoimmune or autoimmune-correlated parameters were investigatedin 91 women with unexplained recurrent abortions (3 consecutive,spontaneous abortions) and 89 fertile control women. Five parameterswere seen significantly more frequently in 19 women with atleast one second trimester miscarriage which had been associatedwith severe intrauterine growth regardation (IUGR), than incontrols. Seventeen of these 19 patients (89%) had at leastone positive autoimmune parameter, compared to 15 of 72 patients(21%) with no second trimester abortions with IUGR (P < 0.0001)and 14 (16%) of the controls (P < 0.0001). No single autoantibodycharacterized patients who exhibited a significant accumulationof autoimmune parameters. These findings may suggest that womenwith recurrent abortions, in whom autoimmunity is thought toplay a role, cannot be identified merely by one laboratory assay,such as that for cardiolipin antibodies, but must be definedby positivity of several criteria. Using our own test panel,preliminary clinical and serological criteria have been setup for the definition of an autoimmune-associated recurrentabortion condition. Twenty-three per cent of the patients inour material fulfilled these criteria, and seven out of nineof these women (78%) have to date been treated successfullywith heparin/aspirin during pregnancy.     

Granulocyte turnover in the feline intestine

The objective of this study was to determine the turnover rate of the extravascular pool of granulocytes in different regions of the feline gastrointestinal tract. Leukocyte emigration from the vasculature was prevented over a 48-h period by repeated intravenous injections of a monoclonal antibody (MAb IB₄) directed against the leukocyte adhesion glycoprotein complex CD11/CD18. Tissue-associated myeloperoxidase (MPO) activity was used to monitor the total tissue granulocyte pool at 0.5, 12, 24, and 48 h after MAb IB₄ administration. The mucosal layer of the duodenum, jejunum, ileum, and colon exhibited different kinetics of granulocyte clearance, with average life-spans (t1/2) ranging between 6.9 (colon) and 10.4 h (duodenum). Granulocyte clearance rates of 0.5 × 10⁶ and 2.4 x 10⁶ cells/h/g tissue were estimated (from measured values oft1/2 and tissue granulocyte pool) for the small bowel and colonie mucosae, respectively. The submucosal layer of the intestine exhibited a biphasic reduction in tissue MPO activity following immunoneutralization of CD11/CD18, with an initialt1/2 0.5 h followed by at1/2 of 36–60 h. The initial rapid decline in tissue MPO suggests that a significant fraction of granulocytes in the submucosa is localized in a readily exchangeable pool (e.g., marginated cells within the vasculature). The results of this study indicate that the average life-span of resident granulocytes varies significantly between different regions of the gastrointestinal tract, with the intestinal mucosa exhibiting at1/2 comparable to that previously reported for circulating feline neutrophils (R 8 h).     

Sulfonylurea-sensitive K+ channels and their probable role for the membrane potential of mouse motor nerve endings

We studied the effect of the K_ATP channel blockers tolbutamide and glibenclamide on presynaptic membrane currents in the mouse M. triangularis sterni preparation using the perineural recording technique. Both sulfonylureas blocked part of the fast K⁺ component within 2 min after application. The block was much more pronounced under glucose-free conditions and was completelyreversible by washing. Addition of glucose to glucose-free bath solution also reduced the K⁺ component. A further effect of the sulfonylureas was observed under glucose-free conditions. With a delay of 5 to 10 min, the nodal Na⁺ component began to diminish and disappeared within 30 min. This was associated with a dramatic increase in spontaneous quantal transmitter release suggesting that the block of sulfonylurea-sensitive K⁺ channels causes depolarization of motor nerve terminals and fibres thus inactivating Na⁺ channels. Tetraethylammonium (TEA) which blocks ATP-dependent K⁺ channels in high concentrations caused, under glucose-free conditions, the same delayed effect as the sulfonylureas. This delayed effect was fully reversible by washing with glucose-containing, but not with glucose-free solution. Our findings strongly suggest that K_ATP channels exist in mammalian motor nerve endings and that under hypoglycemic conditions these channels open and become essential for the maintenance of the membrane potential.