The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression

The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.     

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

Background  

Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites.     

Lability of fat stores in peripubertal wild house mice

Summary Traditionally, the adaptive value of mammalian white fat stores is considered in relation to longterm needs such as providing protection against the vagaries of winter or signalling the reproductive system when energy reserves are sufficient to risk pregnancy. As shown here, the fat stores of young house mice could not serve such needs. Despite prolonged acclimation and excess nesting material, food deprivation at 10°C significantly lowered the fat stores of peripubertal female house mice in only 12 h, and would exhaust them in 30 h. Even close to thermoneutrality (24°C) the calculated time to exhaustion was only 70 h. The fat stores of a young house mouse are obviously too meager to offer any meaningful protection over a winter of several months duration, or even over a 5–6-week cycle of pregnancy and lactation. Furthermore, in a wild habitat where food availability and ambient temperature can vary rapidly and greatly, such fat stores would be too labile to effectively coordinate puberty with somatic development.     

Genetic Characteristics of Restored Elk Populations in Kentucky

Translocations are a common management practice to restore or augment populations. Understanding the genetic consequences of translocation efforts is important for the long-term health of restored populations. The restoration of elk (Cervus canadensis) to Kentucky, USA, included source stocks from 6 western states, which were released at 8 sites in southeastern Kentucky during 1997–2002. We assessed genetic diversity in restored herds and compared genetic similarity to source stocks based on 15 microsatellite DNA loci. Genetic variation in the restored populations was comparable to source stocks ( allelic richness = 3.52 and 3.50; expected heterozygosity = 0.665 and 0.661 for restored and source, respectively). Genetic differentiation among all source and restored populations ranged from 0.000 to 0.065 for pairwise F_ST and 0.034 to 0.161 for pairwise Nei's D_A. Pairwise genetic differentiation and Bayesian clustering revealed that stocks from Utah and North Dakota, USA, contributed most to restored populations. Other western stocks appeared less successful and were not detected with our data, though our sampling was not exhaustive. We also inferred natural movements of elk among release sites by the presence of multiple genetic stocks. The success of the elk restoration effort in Kentucky may be due, in part, to the large number of elk (n = 1,548), repeated releases, and use of diverse source stocks. Future restoration efforts for elk in the eastern United States should consider the use of multiple stock sources and a large number of individuals. In addition, preservation of genetic samples of founder stock will enable detailed monitoring in the future. © 2020 The Authors. The Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.     

The Human GCAP1 and GCAP2 Genes Are Arranged in a Tail-to-Tail Array on the Short Arm of Chromosome 6 (p21.1)

GCAP1 and GCAP2 are related Ca²⁺-binding proteins that activate photoreceptor guanylate cyclase(s). We showed previously that the human GCAP1 gene, consisting of four exons, is located at 6p21.1 (locus designation GUCA). To identify the chromosomal location of the GCAP2 gene, we first cloned its cDNA and determined its intron–exon distribution by PCR analysis. The results show that the introns of the GCAP2 gene are positioned exactly as in the GCAP1 gene and are nearly double in size. Sequence similarity between the two genes, however, is limited to portions of exons 1 and 2. The GCAP1 and GCAP2 genes are transcribed into single mRNA species (1.7 and 2.2 kb, respectively) and are detectable only in the retina by Northern blotting. The GCAP2 gene was found by somatic human–hamster hybrid panel analysis and FISH to reside at GUCA in a region indistinguishable from that of GCAP1. PCR analysis with exon 4-specific primers showed that the genes are in a tail-to-tail array less than 5 kb apart and altogether span less than 20 kb of genomic DNA. The identical gene structures and loci of GCAP1 and GCAP2, and the identical function of the gene products, are consistent with a gene duplication event.     

Rescue of cyclin D1 deficiency by knockin cyclin E.

D-type cyclins and cyclin E represent two very distinct classes of mammalian G1 cyclins. We have generated a mouse strain in which the coding sequences of the cyclin D1 gene (Ccnd1) have been deleted and replaced by those of human cyclin E (CCNE). In the tissues and cells of these mice, the expression pattern of human cyclin E faithfully reproduces that normally associated with mouse cyclin D1. The replacement of cyclin D1 with cyclin E rescues all phenotypic manifestations of cyclin D1 deficiency and restores normal development in cyclin D1-dependent tissues. Thus, cyclin E can functionally replace cyclin D1. Our analyses suggest that cyclin E is the major downstream target of cyclin D1.     

Ultrawideband radiation and pentylenetetrazol-induced convulsions in rats.

New non-ionizing pulsed systems using ultrawideband (UWB) require safety assessment before they can be used by either military or civilian communities. The development of directed energy weaponry intended for use against electronically vulnerable targets, as well as ground-probing radar systems, have used fast-rise-time high-peak-power electromagnetic pulses characteristic of UWB emitters. It has been postulated that these ultrashort pulses might produce electromagnetic transients resulting in tissue damage. Several challenges to this notion have been posed, however. One report found that rats exposed to UWB after receiving a convulsant drug tended toward longer latency to the onset of convulsions than the no-exposure group. Although not statistically significant, the presence of this trend prompted the present study. An ED99 dose of the convulsant pentylenetetrazol (PTZ) or saline was given just before UWB or sham exposure and resultant seizure activity was recorded. The data from the current study show no effect of UWB exposure on PTZ-induced seizure activity, thereby not supporting the tissue damage concerns, at least for the exposure parameters used here.     

Rapid conditional targeted ablation of cells expressing human CD59 in transgenic mice by intermedilysin

Conditional targeted cell ablation is a powerful approach for investigating the pathogenesis of human diseases and in vivo cellular functions. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses human cells exclusively, owing to its receptor specificity for human CD59. We generated two transgenic mouse strains that express human CD59 either on erythrocytes (strain ThCD59(RBC)) or on endothelia (strain ThCD59(END)). Intravenous injection of ILY in ThCD59(RBC) mice induced acute intravascular hemolysis, leading to reduced nitric oxide bioavailability, increased platelet activation and rapid death. In ThCD59(END) mice, ILY induced rapid endothelial damage, leading to acute death and disseminated intravascular coagulation. Additionally, we show that human serum contains ILY-specific neutralizing antibodies not found in any other animal species. Together, these results suggest that this new rapid conditional targeted ILY-mediated cell ablation technique can be used in combination with any available transgenic expression system to study the physiologic role of specific cell populations.