A novel procedure for genotyping of single nucleotide polymorphisms in trisomy with genomic DNA and the invader assay

Individuals with trisomy 21 display complex phenotypes with differing degrees of severity. Numerous reliable methods have been established to diagnose the initial trisomy in these patients, but the identification and characterization of the genetic basis of the phenotypic variation in individuals with trisomy remains challenging. To date, methods that can accurately determine genotypes in trisomic DNA samples are expensive, require specialized equipment and complicated analyses. Here we report proof-of-concept results for an Invader® assay-based genotyping procedure that can determine SNP genotypes in trisomic genomic DNA samples in a simple and cost-effective manner. The procedure requires only two experimental steps: a real-time measurement of the fluorescent Invader® signal and analysis with a specifically designed clustering algorithm. The approach was tested using genomic DNA samples from 23 individuals with trisomy 21, and results were compared to genotypes previously determined with pyrosequencing. Additional assays for 15 SNPs were tested in a set of 21 DNA samples to assess assay performance. Our method successfully identified the correct SNP genotypes for the trisomic genomic DNA samples tested, and thus provides an alternative to determine SNP genotypes in trisomic DNA samples for subsequent association studies in patients with Down syndrome and other trisomies.     

Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.     

Reproducibility of Positive Results for the Detection of Serum Galactomannan by Platelia™ Aspergillus EIA

Galactomannan (GM) was recently included in consensus guidelines as an indirect mycological criterion for the diagnosis of invasive aspergillosis. Currently, there is an enzyme immunoassay available to detect GM in biological samples, the Platelia? Aspergillus EIA. In this study, the reproducibility of positive results obtained using this assay was evaluated using serum samples from neutropenic patients. A trend toward lower values was observed, and 55 %(27/49) of positive results were negative after retesting. A low reproducibility of positive results for the detection of GM in serum was observed.     

The effects of axotomy on electrophysiological properties of B cells of bullfrog sympathetic ganglia conditioned by a previous lesion

In bullfrog sympathetic B cells, axotomy decreases the amplitude and decay time of membrane afterhyperpolarization (AHP) and increases action potential (AP) duration. A second (test) axotomy, 7 days after an initial (conditioning) axotomy, did not amplify these changes. No recovery of AHP amplitude or AP duration occurred by 56 days post-axotomy, but AHP decay time recovered 21 days earlier than after test axotomy alone. Conditioning, previously shown to accelerate regeneration, speeds the return to normal of those membrane properties previously shown to recover after axotomy.     

Comparison of antiprogestin stimulation of uterine prostaglandin synthesis in vitro

Progesterone has an inhibitory effect on prostaglandin synthesis in urine tissue and this effect is reversible with progesterone receptor antagonists. Although antiprogesterone steroids such as RU486 (Mifepristone) are effective at inducing abortion in women they have an improved efficacy when used with exogenous synthetic prostaglandin. In the guinea-pig such antagonists sensitize the uterus but do not result in increased myometrial activity and therefore may not induce endogenous PG synthesis. In this study the effects of antiprogestins on a preparation of rat uterus perifused with progesterone were studied. ZK98 734 caused a rapid and sustained increase in 6-oxoPGF synthesis which rose within the first 90 minutes. This rapid response suggested that some mechanism other than the induction of fresh protein synthesis was involved. A similar increase was not seen with pregnant guinea-pig myometrium/decidua perifused in a similar manner, suggesting that some other mechanism was responsible for the relatively low PG production in pregnancy. However increases in 6-oxoPGF in response to antiprogestins were recorded when pregnant guinea-pig decidua/myometrium was incubated for 4 hours. In these experiments 1 microM ZK98 734 and 1 microM ZK98 299 (Onapristone) gave a 2.7 fold increase in PG production whereas RU486 gave a 1.6 fold increase. Both 1 microM ZK98 734 and 1 microM ZK98 299 also gave a significant increase in PGE production but no increase in PGF was observed. These findings suggest that some antiprogestins might have a better effect on the stimulation of endogenous PG synthesis or on the rate of catabolism of prostanoids.     

Fab is the most efficient format to express functional antibodies by yeast surface display

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.