A micro cell culture method utilizing modified microscope slides for use in fluorescent antibody and enzyme immunoassays

Summary A technique for culturing small quantities of mammalian cells on modified microscope slides is described. The modified microscope slides were Bellco Glass, Inc., toxoplasmosis slides and the cell cultures used were early passage bovine embryonic lung cells and continuous cell lines of porcine and canine origins. The slide cell cultures were either uninfected or infected with selected viruses or the obligate intracellular protozoanEncephalitozoon caniculi for utilization in direct and indirect fluorescent antibody testing or in peroxidase antiperoxidase immunosorbant assays.     

Molecular Manipulations of Extracellular Superoxide Dismutase: Functional Importance for Learning

Extracellular superoxide dismutase (EC-SOD) controls the availability of extracellular superoxide (O ₂ ^- ), which is important for a variety of physiological pathways, including the primary means of inactivating nitric oxide (NO). The role of EC-SOD in neurobehavioral function has been until now unexplored. In the current studies, the phenotypic expression of genotypic alterations of EC-SOD production in mice were characterized for spatial learning and memory. Dramatic impairments in spatial learning in the win-shift 8-arm radial maze were seen in both EC-SOD knockout mice and EC-SOD overexpressing mice. The EC-SOD overexpressing mice were further characterized as having significant deficits in a repeated acquisition task in the radial-arm maze, which permitted the dissociation of long and short-term learning. Long-term learning was significantly impaired by EC-SOD overexpression, whereas short-term learning was not significantly affected by EC-SOD overexpression. NO systems have been shown to be importantly involved in learning and memory. This may be important in the current studies because EC-SOD has primary control over the inactivation of NO. We found that EC-SOD overexpressing mice were resistant to the cognitive effects of L-NAME (N^G-nitro-L-arginine methyl ester hydrochloride), an NO synthase inhibitor. Decreased NO catabolism in these mice may have served to counter the effects of NOS inhibition by L-NAME. The current finding that EC-SOD levels that were either higher or lower than controls impaired learning demonstrates that the proper control of brain extracellular (O ₂ ^- ) may be more vital than merely reduction of brain extracelluar (O ₂ ^- ) in maintaining adequate learning function.     

Electrophoretic variation of avian rotavirus RNA in polyacrylamide gels

Five different sorts of RNA profiles (electropherogroups 1 to 5) were recognised when avian rotavirus RNAs were electrophoresed in polyacrylamide gels. These could be distinguished from the RNAs of mammalian group A, B and C rotaviruses. Viruses belonging to electropherogroups 1, 2/3 and 4 were detected in chickens and viruses belonging to electropherogroups 1, 2, 3 and 5 were detected in turkeys. A minimum of three electropherogroups was recognised in each of four longitudinal surveys of broiler chicken flocks. On examination of rotaviruses from different clinical outbreaks of diarrhoea and from different longitudinal surveys minor differences involving almost all the RNA segments were observed when rotaviruses from the same electropherogroup were compared. Detailed RNA analysis in two of the surveys detected up to three electropherotypes within each electropherogroup.     

Genotype effects and epistasis in type 1 diabetes and HLA-DQ trans dimer associations with disease

Alleles of HLA class II genes DQB1, DQA1, and DRB1 in the MHC region are major determinants of genetic predisposition to type 1 diabetes (T1D). Several alleles of each of these three loci are associated with susceptibility or protection from disease. In addition, relative risks for some DR-DQ genotypes are not simply the sum or product of the single haplotype relative risks. For example, the risk of the DRB1*03-DQB1*02/DRB1*0401-DQB1*0302 genotype is often found to be higher than for the individual DRB1*03-DQB1*02 and DRB1*0401-DQB1*0302 homozygous genotypes. It has been hypothesized that this synergy or epistasis occurs through formation of highly susceptible trans-encoded HLA-DQ(alpha 1, beta 1) heterodimers. Here, we evaluated this hypothesis by estimating the disease associations of the range of DR-DQ genotypes and their inferred dimers in a large collection of nuclear families. We determined whether the risk of haplotypes in DRB1*0401-DQB1*0302-positive genotypes relative to the DRB1*03-DQB1*02-positive genotypes is different from that of DRB1*01-DQB1*0501, which we used as a baseline reference. Several haplotypes showed a different risk compared to DRB1*01-DQB1*0501, which correlated with their ability to form certain trans-encoded DQ dimers. This result provides new evidence for the potential importance of trans-encoded HLA DQ molecules in the determination of HLA-associated risk in T1D.     

Peroxisomal proliferator activated receptor-γ deficiency in a Canadian kindred with familial partial lipodystrophy type 3 (FPLD3)

Background  

Familial partial lipodystrophy (Dunnigan) type 3 (FPLD3, Mendelian Inheritance in Man [MIM] 604367) results from heterozygous mutations in PPARG encoding peroxisomal proliferator-activated receptor-γ. Both dominant-negative and haploinsufficiency mechanisms have been suggested for this condition.     

The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro

Streptococcus pneumoniae culture filtrates and pneumolysin both slow human ciliary beating and damage respiratory epithelium in vitro. A polyclonal pneumolysin antibody bound to sepharose beads removed pneumolysin from culture filtrates and showed that pneumolysin alone was responsible for the effects on epithelium. In a 48-h organ culture pneumolysin caused ciliary slowing and epithelial disruption in a dose-dependent manner down to 5 ng/ml. Comparison of the ciliary slowing activity and pneumolysin concentration in filtrates in a continuous broth culture showed a maximal effect at 16 h (pneumolysin 7.5 micrograms/ml). Later the activity decreased while the pneumolysin concentration increased (8.8 micrograms/ml). This loss of activity was prevented by neutralisation of the acid pH of the culture medium. Eight different culture filtrates produced significant (P less than 0.05) ciliary slowing which correlated (r = 0.95) with simultaneously measured haemolytic (pneumolysin) activity. Substitution of tryptophan (position 433) by phenylalanine reduced the haemolytic and ciliary slowing activity of pneumolysin, but did not affect its ability to activate complement. There was no correlation between the ciliary slowing produced by the culture filtrate and that produced by the autolysate of a particular strain, nor between ciliary slowing and the extent of autolysis or the serotype of the strain.     

A new selective differential medium for isolation ofStenotrophomonas maltophilia

A new selective differential medium for the isolation ofStenotrophomonas (formerlyXanthomonas) maltophilia was developed. The medium, VIA agar, contained vancomycin, imipenem, and amphotericin B as selective agents and incorporated a mannitol/bromothymol blue indicator system. Compared withXanthomonas maltophilia Selective Medium (XMSM), VIA agar was less inhibitory toStenotrophomonas maltophilia and was more selective than XMSM in preventing the growth of unwanted bacteria from contaminated specimens. Although vancomycin-resistant strains ofEnterococcus faecium may grow on VIA agar, these can be easily distinguished fromStenotrophomonas maltophilia because of mannitol fermentation.