Molecular beacon genotyping for globoid cell leukodystrophy from hair roots in the twitcher mouse and rhesus macaque.

Rapid and accurate genotype determination is ideal for the maintenance of breeding colonies of laboratory animal models of genetic disease. The rhesus macaque and murine (twitcher) models of globoid cell leukodystrophy have a dinucleotide deletion or single nucleotide substitution, respectively, which abolish ceramide beta-galactosidase activity and are authentic models of Krabbe disease. We report a molecular beacon PCR assay for each species which allows unambiguous determination of the genotype in under 4h. The assay works reliably with DNA extracted from hair roots using Chelex-100 in a 20 min, 100 degrees C incubation. We demonstrate that genotyping from hair roots is a preferred alternative to collecting blood or tissue for DNA extraction because it reduces animal distress, uses an inexpensive reagent, and is simpler and faster. Following amplification on a standard thermocycler with a 96-well plate format, these molecular beacon assays can be read on a standard laboratory fluorescent plate reader, eliminating the need to use a real-time thermocycler or to open the plate for subsequent restriction enzyme digestion and gel electrophoresis. The multiplexed ratio of fluorescence from wild-type- and mutant-specific beacons reporting at 560 nm and 535 nm wavelengths is distinct for each genotype.

The role of soluble tumor necrosis factor receptor types I and II and tumor necrosis factor-alpha in malaria during pregnancy.

In a prospective study of rhesus monkeys inoculated with Plasmodium coatneyi or saline on an infection/gestational timeline, we determined the serum levels of tumor necrosis factor-alpha (TNF-alpha), soluble tumor necrosis factor receptor type I (sTNFR-I), and soluble tumor necrosis factor receptor type II (sTNFR-II) in peripheral blood throughout primigravid pregnancy, malaria infection, and a combination of the two. Our goal was to determine the association between levels of TNF-alpha and of its 2 soluble receptors and the course of pregnancy and/or malaria and infant outcome. We found that any detectable level of TNF-alpha was always associated with fetal death and that the sTNFRs may be important for fetal protection, possibly through neutralizing the toxic effects of TNF-alpha. Our findings also showed that increased levels of sTNFR-II were associated specifically with malaria and not with normal pregnancy or even pregnancy with low birth weight due to other causes. In contrast, increases in sTNFR-I levels during the later half of normal pregnancies indicate that sTNFR-I may be important in regulating TNF-alpha levels in preparation for normal labor and delivery.

Identification of bovine and novel interferon-tau alleles in the American plains bison (Bison bison) by analysis of hybrid cattle x bison blastocysts.

The objective of this study was to generate bison x cattle hybrid embryos by in vitro fertilization, to assess their developmental potential, to determine the pattern of secretion of the embryonic signaling molecule interferon-tau (IFN-tau), and to identify novel IFN-tau mRNA polymorphism in the American plains bison. A total of 600 bovine oocytes were inseminated with frozen-thawed bison semen. Of these, 40.7% cleaved and 14.8% proceeded to the blastocyst stage. Individual blastocysts were cultured on a basement membrane (Matrigel) and their ability to attach and form outgrowths was monitored. A total of 36 blastocysts were cultured of which 22 formed outgrowths. During individual culture, medium samples were collected and their IFN-tau concentration was measured. On day 6 after onset of individual culture, attached outgrowths produced significantly more IFN-tau than unattached viable or degenerate blastocysts. At this time, female conceptuses also produced significantly more IFN-tau than their male cohorts. However, by day 12 this difference had disappeared. Total mRNA was extracted from three individual outgrowths and analyzed by RT-PCR. Subsequent sequencing of 28 clones showed several known bovine IFN-tau sequences as well as two novel sequences termed bisIFN-tau1 and 2. To determine the origin of these, DNA was extracted from bison semen and analyzed by PCR. One bovine IFN-tau sequence (bovIFN-tau1d) as well as bisIFN-tau2 and a third novel sequence bisIFN-tau3 were detected. This study demonstrates the feasibility of using hybrid embryos for the analysis of developmentally regulated gene expression in species where embryos may not be available.

SIV-induced activation of the blood-brain barrier requires cell-associated virus and is not restricted to endothelial cell activation.

It has never been determined if activation of the blood-brain barrier (BBB) during simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection is a function of high levels of circulating virus or if the virus has to be within a cell capable of crossing the BBB to activate it. In vitro models of the BBB are becoming recognized as an acceptable method for determining the cellular events associated with HIV neuroinvasion. Cell free virus (when added in the physiologically relevant lumen) although capable of activating the endothelial cells of our in vitro BBB did not activate astrocytes beneath. SIVmac251-infected CEMx174 cells, however, were capable of activating both components of the BBB model. Here we demonstrate that an in vitro model of the BBB can be activated in a physiologically relevant manner, that SIV requires to be cell-associated and that endothelial cells of the BBB are not the only components that are activated during SIV neuroinvasion.

Interferon-tau in bovine blastocysts following parthenogenetic activation of oocytes: pattern of secretion and polymorphism in expressed mRNA sequences.

A series of experiments were conducted to examine the pattern of production and secretion of interferon-tau (IFN-tau) by blastocysts following parthenogenetic activation of bovine oocytes. In the first experiment, 36.8, 24.1, and 33.2% of IVF-derived and parthenogenetically activated oocytes cultured in the presence or absence of a monolayer of buffalo rat liver cells, respectively, reached the blastocyst stage. Following individual culture of blastocysts, IFN-tau concentration in medium droplets was similar among the three groups, although IVF-derived blastocysts contained significantly more cells. In the second experiment, 156 IVF-derived blastocysts were sexed by PCR with 75 and 81, respectively, being male and female. IFN-tau secretion of these was compared to that of 70 parthenogenetic blastocysts. Female and parthenogenetic blastocysts produced significantly more IFN-tau than their male counterparts. In the third experiment, the ability of hatched blastocysts to form outgrowths and the pattern of their IFN-tau secretion were examined. Of the 48 IVF-derived blastocysts, 44 formed outgrowths compared to 41 of the 42 hatched parthenotes. Parthenogenetic outgrowths were significantly larger after 7 days, but this difference had disappeared after 14 days. IFN-tau secretion did not differ between the two groups. Lastly, sequence analyses of expressed mRNA from individual parthenogenetic blastocyst outgrowths showed four different transcript types which, based on their predicted amino acid sequence, belong to two subgroups, IFN-tau1 and IFN-tau3. In addition, one new transcript sequence was identified, encoding a new protein isoform.

G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme.

G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4. Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype. The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation. The results suggest that G350 contributes to Mg(2+) binding at helix P4 of RNase P RNA.