Selective antihypertensive dihydropyridines lower Aβ accumulation by targeting both the production and the clearance of Aβ across the blood-brain barrier

Several large population-based or clinical trial studies have suggested that certain dihydropyridine (DHP) L-type calcium channel blockers (CCBs) used for the treatment of hypertension may confer protection against the development of Alzheimer disease (AD). However, other studies with drugs of the same class have shown no beneficial clinical effects. To determine whether certain DHPs are able to impact underlying disease processes in AD (specifically the accumulation of the Alzheimer Aβ peptide), we investigated the effect of several antihypertensive DHPs and non-DHP CCBs on Aβ production. Among the antihypertensive DHPs tested, a few, including nilvadipine, nitrendipine and amlodipine inhibited Aβ production in vitro, whereas others had no effect or raised Aβ levels. In vivo, nilvadipine and nitrendipine acutely reduced brain Aβ levels in a transgenic mouse model of AD (Tg PS1/APPsw) and improved Aβ clearance across the blood-brain barrier (BBB), whereas amlodipine and nifedipine were ineffective showing that the Aβ-lowering activity of the DHPs is independent of their antihypertensive activity. Chronic oral treatment with nilvadipine decreased Aβ burden in the brains of Tg APPsw (Tg2576) and Tg PS1/APPsw mice, and also improved learning abilities and spatial memory. Our data suggest that the clinical benefit conferred by certain antihypertensive DHPs against AD is unrelated to their antihypertensive activity, but rely on their ability to lower brain Aβ accumulation by affecting both Aβ production and Aβ clearance across the BBB.     

Coordinate and independent regulation of αB-crystallin and HSP27 expression in response to physiological stress

α-Crystallins share structural and functional properties with the stress protein hsp27. These polypeptides are expressed at low constitutive levels in many tissues including brain, and αB-crystallin and hsp27 can accumulate in central nervous system glia in a variety of neurological conditions. We report here that heat shock and exposure to transition metals result in an increase in the steady state mRNA level of αB-crystallin and hsp27 in primary cultures of rat forebrain astrocytes. Both exposure to tumour necrosis factor-α and hypertonic conditions result in αB-crystallin mRNA accumulation but no change in the hsp27 mRNA level. Under some of these conditions increased synthesis and accumulation of αB-crystallin and hsp27 protein are also evident. We are unable to detect αA-crystallin mRNA in resting or stressed astrocytes. A novel phenomenon involving a transitory change in stress protein mRNA mobility in Northern blots during induction is reported, which is stress type and cell type independent. The results demonstrate multiple stress regulation of αB-crystallin and hsp27 in cultured astrocytes, suggesting that they can legitimately be regarded as stress proteins in the central nervous system. © 1994 wiley-Liss, Inc.     

RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: Consequences on lignans and neolignans accumulation

RNAi technology was applied to down regulate LuPLR1 gene expression in flax (Linum usitatissimum L.) seeds. This gene encodes a pinoresinol lariciresinol reductase responsible for the synthesis of (+)-secoisolariciresinol diglucoside (SDG), the major lignan accumulated in the seed coat. If flax lignans biological properties and health benefits are well documented their roles in planta remain unclear. This loss of function strategy was developed to better understand the implication of the PLR1 enzyme in the lignan biosynthetic pathway and to provide new insights on the functions of these compounds. RNAi plants generated exhibited LuPLR1 gene silencing as demonstrated by quantitative RT-PCR experiments and the failed to accumulate SDG. The accumulation of pinoresinol the substrate of the PLR1 enzyme under its diglucosylated form (PDG) was increased in transgenic seeds but did not compensate the overall loss of SDG. The monolignol flux was also deviated through the synthesis of 8-5′ linked neolignans dehydrodiconiferyl alcohol glucoside (DCG) and dihydro-dehydrodiconiferyl alcohol glucoside (DDCG) which were observed for the first time in flax seeds.     

Phosphorylation of smg p21B/rap1B p21 by cyclic GMP-dependent protein kinase.

smg p21B/rap1B p21, a member of ras p21-like small GTP-binding protein superfamily, has been shown to be phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). We show here that this protein was also phosphorylated by cyclic GMP-dependent protein kinase (protein kinase G) in a cell-free system. The same serine residue (Ser179) in the C-terminal region was phosphorylated by both protein kinases G and A. The Km and Vmax values of smg p21B for protein kinase G were 5 x 10(-7) M and 4 x 10(-9) mol/min/mg, and those values for protein kinase A were 1 x 10(-7) M and 3 x 10(-8) mol/min/mg.     

Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilms

A microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM (CAM). Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 μm/min. An enzyme-based antimicrobial formulation led to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in the removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.     

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.     

NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22^phox and gp91^phox, as well as the Ca²⁺ concentration-responsive gp91^phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22^phox was not detectable, both gp91^phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22^phox or gp91^phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death. superoxide anion; diphenylene iodonium; p22^phox; gp91^phox; adenosine 3',5'-cyclic monophosphate response element; caspases     

Analysis of the structural basis of specificity of inhibition of the Abl kinase by STI571

STI571, a selective inhibitor of Bcr-Abl, has been a successful therapeutic agent in clinical trials for chronic myelogenous leukemia. Chronic phase chronic myelogenous leukemia patients treated with STI571 have durable responses; however, most responding blast phase patients relapse despite continued therapy. Co-crystallization studies of Abl kinase and an STI571-related compound identify specific amino acid residues as critical to STI571 binding, one of which, T315, has been characterized as an acquired Thr to Ile mutation in relapsed patients. Other studies, however, suggest that mutations other than these predicted contact points are capable of conferring STI571 resistance in relapsed patients. Using a variety of models of STI571 binding to the Abl kinase, we have performed an extensive mutational analysis of sites that might alter the sensitivity of the Abl kinase to STI571. Although mutation of many of the predicted contact points between Abl and STI571 result in a kinase-inactive protein, additional mutations that render the Abl kinase less sensitive to STI571 demonstrate a broad range of possibilities for clinical resistance that are now becoming evident.     

Gas channels for NH(3): proteins from hyperthermophiles complement an Escherichia coli mutant

Ammonium transport (Amt) proteins appear to be bidirectional channels for NH(3). The amt genes of the hyperthermophiles Aquifex aeolicus and Methanococcus jannaschii complement enteric amtB mutants for growth at 25 nM NH(3) at 37 degrees C. To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium. Despite low expression levels, His-tagged Aquifex Amt could be purified by heating and nickel chelate affinity chromatography. It could be studied genetically in Escherichia coli.