Differential contribution of the three Aph1 genes to gamma-secretase activity in vivo

Gamma-secretase is the protease responsible for amyloid beta peptide release and is needed for Notch, N-Cadherin, and possibly other signaling pathways. The protease complex consists of at least four subunits, i.e., Presenilin, Aph1, Pen2, and Nicastrin. Two different genes encode Aph1A and Aph1B in man. A duplication of Aph1B in rodents has given rise to a third gene, Aph1C. Different mixes of gamma-secretase subunits assemble in at least four human and six rodent complexes but it is not known whether they have different activities in vivo. We report here the inactivation of the three Aph1 genes in mice. Aph1A-/- embryos show a lethal phenotype characterized by angiogenesis defects in the yolk sac, neuronal tube malformations, and mild somitogenesis defects. Aph1B-/- or C-/- or the combined Aph1BC-/- mice (which can be considered as a model for total Aph1B loss in human) survive into adulthood. However, Aph1BC-/- deficiency causes a mild but significant reduction in amyloid beta percursor protein processing in selective regions of the adult brain. We conclude that the biochemical and physiological repercussions of genetically reducing gamma-secretase activity via the different Aph1 components are quite divergent and tissue specific. Our work provides in vivo evidence for the concept that different gamma-secretase complexes may exert different biological functions. In the context of Alzheimer's disease therapy, this implies the theoretical possibility that targeting specific gamma-secretase subunit combinations could yield less toxic drugs than the currently available general inhibitors of gamma-secretase activity.     

Left ventricular diastolic function in relation to the urinary proteome: A proof-of-concept study in a general population

Background

In previous studies, we identified two urinary proteomic classifiers, termed HF1 and HF2, which discriminated subclinical diastolic left ventricular (LV) dysfunction from normal. HF1 and HF2 combine information from 85 and 671 urinary peptides, mainly up- or down-regulated collagen fragments. We sought to validate these classifiers in a population study.

Methods

In 745 people randomly recruited from a Flemish population (49.8 years; 51.3% women), we measured early and late diastolic peak velocities of mitral inflow (E and A) and mitral annular velocities (e' and a') by conventional and tissue Doppler echocardiography, and the urinary proteome by capillary electrophoresis coupled with mass spectrometry.

Results

In the analyses adjusted for sex, age, body mass index, blood pressure, heart rate, LV mass index and intake of medications, we expressed effect sizes per 1-SD increment in the classifiers. HF1 was associated with 0.204 cm/s lower e' peak velocity (95% confidence interval, 0.057–0.351; p = 0.007) and 0.145 higher E/e' ratio (0.023–0.268; p = 0.020), while HF2 was associated with a 0.174 higher E/e' ratio (0.046–0.302; p = 0.008). According to published definitions, 67 (9.0%) participants had impaired LV relaxation and 96 (12.9%) had elevated LV filling pressure. The odds of impaired relaxation associated with HF1 was 1.38 (1.01–1.88; p = 0.043) and that of increased LV filling pressure associated with HF2 was 1.38 (1.00–1.90; p = 0.052).

Conclusions

In a general population, the urinary proteome correlated with diastolic LV dysfunction, proving its utility for early diagnosis of this condition.     

CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist.

CCR5 was first characterized as a receptor for MIP-1alpha, MIP-1beta, and RANTES, and was rapidly shown to be the main coreceptor for M-tropic human immunodeficiency virus (HIV)-1 strains and simian immunodeficiency virus (SIV). Chemokines constitute a rapidly growing family of proteins and receptor-chemokine interactions are known to be promiscuous and redundant. We have therefore tested whether other CC-chemokines could bind to and activate CCR5. All CC-chemokines currently available were tested for their ability to compete with [(125)I]-MIP-1beta binding on a stable cell line expressing recombinant CCR5, and/or to induce a functional response in these cells. We found that in addition to MIP-1beta, MIP-1alpha, and RANTES, five other CC-chemokines could compete for [(125)I]-MIP-1beta binding: MCP-2, MCP-3, MCP-4, MCP-1, and eotaxin binding was characterized by IC(50) values of 0.22, 2.14, 5.89, 29.9, and 21.7 nmol/L, respectively. Among these ligands, MCP-3 had the remarkable property of binding CCR5 with high affinity without eliciting a functional response, MCP-3 could also inhibit the activation of CCR5 by MIP-1beta and may therefore be considered as a natural antagonist for CCR5. It was unable to induce significant endocytosis of the receptor. Chemokines that could compete with high affinity for MIP-1beta binding could also compete for monomeric gp120 binding, although with variable potencies; maximal gp120 binding inhibition was 80% for MCP-2, but only 30% for MIP-1beta. MCP-3 could compete efficiently for gp120 binding but was, however, found to be a weak inhibitor of HIV infection, probably as a consequence of its inability to downregulate the receptor.     

Study of the stability of polymyxins B(1), E(1) and E(2) in aqueous solution using liquid chromatography and mass spectrometry

Polymyxins B(1), E(1) (colistin A) and E(2) (colistin B) were subjected to degradation in aqueous solutions of different pH values (1.4, 3.4, 5.4 and 7.4) and at different temperatures (37, 50 and 60 degrees C) in order to investigate the characteristics of decomposition. The progress of decomposition was followed by reversed-phase liquid chromatography on YMC-Pack Pro, C-18 stationary phase. The degradation curves showed (pseudo) first order kinetics. The pH-rate profiles indicate that colistin is more susceptible to degradation in solutions of pH above 5 and is more stable in acidic media. The degradation of polymyxin B(1) was most rapid at pH 7.4. Qualitative analysis of the degradation products by LC/MS reveals that racemization is the major mechanism of degradation in both acidic and neutral media.     

The antiplasmodial activity of isolates from Ajuga remota

Ajuga remota is the most frequently used medicinal herb for malaria treatment in Kenya. Its two known isolates ajugarin-1 (1) and ergosterol-5,8-endoperoxide (3) and a new isolate 8-O-acetylharpagide (2) were evaluated for their in vitro antiplasmodial activity. Ajugarin-1 was moderately active, with an IC(50) of 23.0 +/- 3.0 microM, as compared to chloroquine (IC(50) = 0.041 +/- 0.003 microM) against the chloroquine-sensitive (FCA 20/GHA) strain of Plasmodium falciparum. Ergosterol-5,8-endoperoxide was about 3x as potent (IC(50) = 8.2 +/- 1.1 microM), while 8-O-acetylharpagide, whose structure was established by spectroscopic evidence, was inactive. Both ajugarin-1 and ergosterol-5,8-endoperoxide did not exhibit cytotoxicity against A431 (skin carcinoma) cell line, but 8-O-acetylharpagide was significantly cytotoxic. This iridoid glucoside, which has been formerly isolated from Ajuga decumbens, was identified in A. remota for the first time.