Prion protein gene polymorphisms in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae genome encodes several proteins that, in laboratory strains, can take up a stable, transmissible prion form. In each case, this requires the Asn/Gln-rich prion-forming domain (PrD) of the protein to be intact. In order to further understand the evolutionary significance of this unusual property, we have examined four different prion genes and their corresponding PrDs, from a number of naturally occurring strains of S. cerevisiae. In 4 of the 16 strains studied we identified a new allele of the SUP35 gene (SUP35delta19) that contains a 19-amino-acid deletion within the N-terminal PrD, a deletion that eliminates the prion property of Sup35p. In these strains a second prion gene, RNQ1, was found to be highly polymorphic, with eight different RNQ1 alleles detected in the six diploid strains studied. In contrast, for one other prion gene (URE2) and the sequence of the NEW1 gene encoding a PrD, no significant degree of DNA polymorphism was detected. Analysis of the naturally occurring alleles of RNQ1 and SUP35 indicated that the various polymorphisms identified were associated with DNA tandem repeats (6, 12, 33, 42 or 57 bp) within the coding sequences. The expansion and contraction of DNA repeats within the RNQ1 gene may provide an evolutionary mechanism that can ensure rapid change between the [PRION+] and [prion-] states.     

Neck afferents and muscle sympathetic activity in humans: implications for the vestibulosympathetic reflex

Ray, Chester A., and Keith M. Hume. Neck afferents andmuscle sympathetic activity in humans: implications for the vestibulosympathetic reflex. J. Appl.Physiol. 84(2): 450-453, 1998.We have shownpreviously that head-down neck flexion (HDNF) in humans elicitsincreases in muscle sympathetic nerve activity (MSNA). The purpose ofthis study was to determine the effect of neck muscle afferents onMSNA. We studied this question by measuring MSNA before and after headrotation that would activate neck muscle afferents but not thevestibular system (i.e., no stimulation of the otolith organs orsemicircular canals). After a 3-min baseline period with the head inthe normal erect position, subjects rotated their head to the side(~90°) and maintained this position for 3 min. Head rotation wasperformed by the subjects in both the prone(n = 5) and sitting(n = 6) positions. Head rotation did not elicit changes in MSNA. Average MSNA, expressed asburst frequency and total activity, was 13 ± 1 and 13 ± 1 bursts/min and 146 ± 34 and 132 ± 27 units/min during baselineand head rotation, respectively. There were no significant changes incalf blood flow (2.6 ± 0.3 to 2.5 ± 0.3 ml · 100 ml¹ · min¹;n = 8) and calf vascular resistance(39 ± 4 to 41 ± 4 units; n = 8). Heart rate (64 ± 3 to 66 ± 3 beats/min;P = 0.058) and mean arterial pressure(90 ± 3 to 93 ± 3; P < 0.05)increased slightly during head rotation. Additional neck flexionstudies were performed with subjects lying on their side(n = 5). MSNA, heart rate, and meanarterial pressure were unchanged during this maneuver, which also doesnot engage the vestibular system. HDNF was tested in 9 of the 13 subjects. MSNA was significantly increased by 79 ± 12% (P < 0.001) during HDNF. Thesefindings indicate that neck afferents activated by horizontal neckrotation or flexion in the absence of significant force development donot elicit changes in MSNA. These findings support the concept thatHDNF increases MSNA by the activation of the vestibular system.

     

Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering.     

Detecting consistent common lines in cryo-EM by voting

The single-particle reconstruction problem of electron cryo-microscopy (cryo-EM) is to find the three-dimensional structure of a macromolecule given its two-dimensional noisy projection images at unknown random directions. Ab initio estimates of the 3D structure are often obtained by the “Angular Reconstitution” method, in which a coordinate system is established from three projections, and the orientation of the particle giving rise to each image is deduced from common lines among the images. However, a reliable detection of common lines is difficult due to the low signal-to-noise ratio of the images. In this paper we describe a global self-correcting voting procedure in which all projection images participate to decide the identity of the consistent common lines. The algorithm determines which common line pairs were detected correctly and which are spurious. We show that the voting procedure succeeds at relatively low detection rates and that its performance improves as the number of projection images increases. We demonstrate the algorithm for both simulative and experimental images of the 50S ribosomal subunit.     

Azole-based inhibitors of AKT/PKB for the treatment of cancer

Through a combination of screening and structure-based rational design, we have discovered a series of N¹-(5-(heterocyclyl)-thiazol-2-yl)-3-(4-trifluoromethylphenyl)-1,2-propanediamines that were developed into potent ATP competitive inhibitors of AKT. Studies of linker strand-binding adenine isosteres identified SAR trends in potency and selectivity that were consistent with binding interactions observed in structures of the inhibitors bound to AKT1 and to the counter-screening target PKA. One compound was shown to have acceptable pharmacokinetic properties and to be a potent inhibitor of AKT signaling and of in vivo xenograft tumor growth in a preclinical model of glioblastoma.     

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.     

The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein

The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.