Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.

Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.     

A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.     

Chemical characterization of the regularly arranged surface layer glycoprotein of Clostridium thermosaccharolyticum D120-70

Clostridium thermosaccharolyticum D120-70 possesses as its outermost cell envelope layer a square-arranged array of glycoprotein molecules. SDS/polyacrylamide gel electrophoresis of the purified surface layer showed a broadened band in the molecular mass range of about 115 kDa which, upon periodic acid/Schiff staining, gave a positive reaction. After proteolytic degradation of this material, two glycopeptide fractions were obtained. One- and two-dimensional nuclear magnetic resonance studies, together with methylation analysis and periodate oxidation, were used to determine the structures of the polysaccharide portions of these glycopeptides. The combined chemical and spectroscopic evidence suggests the following structures: (formula; see text).     

Morphological gradients in the starling basilar papilla

The starling cochlea was studied with TEM at four locations along the basilar papilla to investigate gradients in morphological features over the papilla's length and width. Hair cell shape changes continuously from neural to abneural and from basal to apical. Unlike the situation in mammals, there are no distinct populations of hair cells; the previously described types (tall hair cells and short hair cells) are merely extremes in a continuum. Contacts between THC are a normal feature. Except at the base of the papilla, SHC have very large cuticular plates, suggesting a micromechanical function for these cells. In contrast to the THC, the SHC normally completely lack afferent innervation; this indicates that their function is restricted to within the basilar papilla itself. © 1992 Wiley-Liss, Inc.     

Identification and design of a novel series of MGAT2 inhibitors

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to na?ve, p <0.01) in plasma triacylglycerol (TAG) concentration.     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Morphological Parameters Associated with Ruptured Posterior Communicating Aneurysms

The rupture risk of unruptured intracranial aneurysms is known to be dependent on the size of the aneurysm. However, the association of morphological characteristics with ruptured aneurysms has not been established in a systematic and location specific manner for the most common aneurysm locations. We evaluated posterior communicating artery (PCoA) aneurysms for morphological parameters associated with aneurysm rupture in that location. CT angiograms were evaluated to generate 3-D models of the aneurysms and surrounding vasculature. Univariate and multivariate analyses were performed to evaluate morphological parameters including aneurysm volume, aspect ratio, size ratio, distance to ICA bifurcation, aneurysm angle, vessel angles, flow angles, and vessel-to-vessel angles. From 2005–2012, 148 PCoA aneurysms were treated in a single institution. Preoperative CTAs from 63 patients (40 ruptured, 23 unruptured) were available and analyzed. Multivariate logistic regression revealed that smaller volume (p = 0.011), larger aneurysm neck diameter (0.048), and shorter ICA bifurcation to aneurysm distance (p = 0.005) were the most strongly associated with aneurysm rupture after adjusting for all other clinical and morphological variables. Multivariate subgroup analysis for patients with visualized PCoA demonstrated that larger neck diameter (p = 0.018) and shorter ICA bifurcation to aneurysm distance (p = 0.011) were significantly associated with rupture. Intracerebral hemorrhage was associated with smaller volume, larger maximum height, and smaller aneurysm angle, in addition to lateral projection, male sex, and lack of hypertension. We found that shorter ICA bifurcation to aneurysm distance is significantly associated with PCoA aneurysm rupture. This is a new physically intuitive parameter that can be measured easily and therefore be readily applied in clinical practice to aid in the evaluation of patients with PCoA aneurysms.     

Peripheral and central nervous responses evoked by small water movements in a cephalopod

Potentials were recorded from the epidermal head lines and from the CNS of young cuttlefish, Sepia officinalis, in response to weak water movements. 1. Within the test range 0.5-400 Hz a sinusoidal water movement elicits up to 4 components of response if the electrode is placed on a headline: (i) a positive phasic ON response; (ii) a tonic frequency-following microphonic response; (iii) a slow negative OFF response; and (iv) compound nerve impulses. 2. The amplitude of both the ON wave and the microphonic potential depends on stimulus frequency, stimulus amplitude and stimulus rise time. Frequencies around 100 Hz and short rise times are most effective in eliciting strong potentials. The minimal threshold was 0.06 microns peak-to-peak water displacement at 100 Hz (18.8 microns/s as velocity). 3. Change of direction of tangential sphere movement (parallel vs. across the head lines) has only a small effect on the microphonic and the summed nerve potentials. 4. Frequency and/or amplitude modulations of a carrier stimulus elicit responses at the onset and offset of the modulation and marked changes in the tonic microphonic response. 5. Evoked potentials can be recorded from the brain while stimulating the epidermal lines with weak water movements. The brain potentials differ in several aspects from the potentials of the head lines and show little or no onset or offset wave at the transitions of a frequency and amplitude modulation.