Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues

Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology.     

Characterization of an Endoglucanase from Pseudomonas fluorescens subsp. cellulosa Produced in Escherichia coli and Regulation of the Expression of Its Cloned Gene

Several enzymatic properties of an endoglucanase produced in Escherichia coli by a gene from Pseudomonas fluorescens subsp. cellulosa were investigated. Gel filtration revealed a single peak of M_r 36,000 with endoglucanase activity. The pH optimum of the enzyme was 7.0. Carboxymethyl cellulose and barley β-glucan (mixed β-1,3 and 1,4 linkages) were good substrates, but not laminarin (β-1,3 linkages), amylose, filter paper, microcrystalline cellulose (Avicel), or cellotriose. The mode of action was typical of an “endo”-acting enzyme. Taken together, these properties do not correspond to those of any of the endoglucanases described in P. fluorescens subsp. cellulosa. Consequently, the gene was designated egIX. The enzyme was sensitive to end-product inhibition by cellobiose but was only moderately inhibited by glucose. The enzyme was formed constitutively in E. coli throughout the growth phase. Urea had no effect on endoglucanase synthesis, but glucose acted as a catabolite repressor. The formation of the enzyme in E. coli was partially dependent on cyclic AMP.     

Spatial Variation of Soil CO<Subscript>2</Subscript>, CH<Subscript>4</Subscript> and N<Subscript>2</Subscript>O Fluxes Across Topographical Positions in Tropical Forests of the Guiana Shield

The spatial variation of soil greenhouse gas fluxes (GHG; carbon dioxide—CO₂, methane—CH₄ and nitrous oxide—N₂O) remains poorly understood in highly complex ecosystems such as tropical forests. We used 240 individual flux measurements of these three GHGs from different soil types, at three topographical positions and in two extreme hydric conditions in the tropical forests of the Guiana Shield (French Guiana, South America) to (1) test the effect of topographical positions on GHG fluxes and (2) identify the soil characteristics driving flux variation in these nutrient-poor tropical soils. Surprisingly, none of the three GHG flux rates differed with topographical position. CO₂ effluxes covaried with soil pH, soil water content (SWC), available nitrogen and total phosphorus. The CH₄ fluxes were best explained by variation in SWC, with soils acting as a sink under drier conditions and as a source under wetter conditions. Unexpectedly, our study areas were generally sinks for N₂O and N₂O fluxes were partly explained by total phosphorus and available nitrogen concentrations. This first study describing the spatial variation of soil fluxes of the three main GHGs measured simultaneously in forests of the Guiana Shield lays the foundation for specific studies of the processes underlying the observed patterns.     

Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli.

Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation.     

Metabolism of D-glucose anomers in rat pancreatic islets exposed to equilibrated D-glucose.

This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.     

Microbiology Study of a Hypersaline Lake in French Somaliland

In a study of a lake having a higher concentration of salts than the Dead Sea, all of the heterotrophic bacteria isolated were aerobes; no strictly anaerobic strains were found. Ninety percent of the strains were euryhalines and ten percent were strict halophiles. The extreme halophiles belonged to the species Halobacterium trapanicum and Halococcus morrhuae.