Computer Simulation of Assembly and Co-operativity of Hexameric AAA ATPases

AAA ATPases form a functionally diverse superfamily of proteins. Most members form homo-hexameric ring complexes, are catalytically active only in the fully assembled state, and show co-operativity among the six subunits. The mutual dependence among the subunits is clearly evidenced by the fact that incorporation of mutated, inactive subunits can decrease the activity of the remaining wild type subunits. For the first time, we develop here models to describe this form of allostery, evaluate them in a simulation study, and test them on experimental data. We show that it is important to consider the assembly reactions in the kinetic model, and to define a formal inhibition scheme. We simulate three inhibition scenarios explicitly, and demonstrate that they result in differing outcomes. Finally, we deduce fitting formulas, and test them on real and simulated data. A non-competitive inhibition formula fitted experimental and simulated data best. To our knowledge, our study is the first one that derives and tests formal allosteric schemes to explain the inhibitory effects of mutant subunits on oligomeric enzymes.     

Dissociable Perceptual Effects of Visual Adaptation

Neurons in the visual cortex are responsive to the presentation of oriented and curved line segments, which are thought to act as primitives for the visual processing of shapes and objects. Prolonged adaptation to such stimuli gives rise to two related perceptual effects: a slow change in the appearance of the adapting stimulus (perceptual drift), and the distortion of subsequently presented test stimuli (adaptational aftereffects). Here we used a psychophysical nulling technique to dissociate and quantify these two classical observations in order to examine their underlying mechanisms and their relationship to one another. In agreement with previous work, we found that during adaptation horizontal and vertical straight lines serve as attractors for perceived orientation and curvature. However, the rate of perceptual drift for different stimuli was not predictive of the corresponding aftereffect magnitudes, indicating that the two perceptual effects are governed by distinct neural processes. Finally, the rate of perceptual drift for curved line segments did not depend on the spatial scale of the stimulus, suggesting that its mechanisms lie outside strictly retinotopic processing stages. These findings provide new evidence that the visual system relies on statistically salient intrinsic reference stimuli for the processing of visual patterns, and point to perceptual drift as an experimental window for studying the mechanisms of visual perception.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.     

Experimental infections of simians with human malaria: attempts to infect Callithrix penicillata with Plasmodium falciparum

After reviewing the use of non-human primates of the Old and New Worlds for human malaria research, we concluded that another experimental animal which is easily available to use and possible to rear indoors is needed. Thus, we studied the susceptibility of the marmoset Callithrix penicillata to Plasmodium falciparum erythrocytic infections. The marmosets received various P. falciparum human isolates, directly from a patient and from continuous cultures. The Palo Alto strain, which has been adapted to the night monkey Aotus trivirgatus and further maintained in the squirrel monkey Saimiri sciureus was also used. In a total of 20 marmosets we performed 31 inoculations, with 10(5) to 10(9) parasites, intraperitoneally, intracardiacly or intravenously. Blood samples from each animal were examined daily up to day 90 post-inoculation. None of the intact marmosets developed patent infections. Four out of 19 C. penicillata, previously splenectomized, showed circulating parasites for up to five days after intravenous inoculation with the Palo Alto strain, becoming negative thereafter. Neither the addition to the simian diet of p-aminobenzoic acid, essential for the parasite metabolism, nor drug-immunosuppression, improved the marmoset susceptibility to P. falciparum.     

Taste systems of the petrosal ganglion of the rat glossopharyngeal nerve

Single unit recordings were taken from sensory ganglion cellsin the petrosal ganglion (PG) of the glossopharyngeal nerveof the rat. These taste units were examined with respect tospontaneous and evoked discharge patterns and responsivenessto a wide variety of chemical compounds, most of natural occurrence.Spontaneous activity patterns, with few exceptions, tended tobe extremely irregular with both bursting (clusters of 2–3spikes) and grouping (large groups of spikes as in evoked discharges).Most interspike interval histograms of spontaneous activitywere multimodal, similar to rat geniculate ganglion (GG) units.Evoked discharges usually displayed grouping of spikes, andlong latencies of onset and persistence of discharge after rinsewere sometimes seen. Little response was shown to nucleotidesor salts. Units responsive to amino acids tended to show largedischarge to only one or two amino acids; and the most responsiveamino acid usually varied from cell to cell. Units responsiveto alkaloids only responded to a few alkaloids with atropineand quinine being the most stimulatory. Units responsive toacids only discharged to a few of the acids tested and oftenacids of low pH elicited no discharge. Saccharin activated unitsresponsive to both sugar and alkaloids. A few units highly responsiveto both sugar and alkaloids were seen. The units were placedinto four clusters on the basis of chemicals activating themand certain neurophysiological characteristics: PG salt units,PG acid units and, tentatively, amino acid (sugar) units andX (alkaloid and alkaloid plus) units. The PG salt units didnot show the exclusive sensitivity to sodium and lithium compoundsas did the GG salt units. The PG acid units could also be differentiatedfrom the GG acid units. The petrosal amino acid and X units,on the other hand, could not be differentiated from similarunits in the rat GG.     

Neurophysiology of geniculate ganglion (facial nerve) taste systems: species comparisons

On the basis of various measures taken from geniculate gangliontaste neurons in four species, it was concluded that withineach species the neurons could be subdivided into distinct functionalgroups. In this report, the neural groups of different specieswere directly compared. Units from all four species were studiedwith a common test series of solutions in addition to otherstimuli. Since these stimuli were presented at the same concentrationsto all species, direct quantitative comparisons across specieswere possible for a wide range of chemical compounds. In addition,the neural groups were compared with respect to spontaneousand evoked activity measures, latency to electrical stimulation,and receptive field characteristics. These neurophysiologicaldata suggest a basic model of four distinct subgroups: acidunits, salt units, amino acid units, and X units.     

Cysteine: Depolarization-Induced Release from Rat Brain In Vitro

Compounds released on depolarization in a Ca2+-dependent manner from rat brain slices were screened to identify candidates for neuroactive substances. Lyophilized superfusates were analyzed by reversed-phase HPLC after derivatization with 9-fluorenyl N-succinimidyl carbonate. One of the compounds that showed an increase of concentration in superfusates in the presence of iodoacetamide was identified as the cysteine (Cys) derivative, S-carboxamidomethylcysteine, by fast atom bombardment mass spectrometry and other methods. This stable Cys derivative originates from endogenous, extracellular Cys. The finding led to a method for quantification of Cys in superfusates by immediate cooling of the superfusates to 0 degrees C and reaction of Cys with N-ethylmaleimide. Depolarization-induced Ca2+-dependent release of Cys was most prominent in the neocortex, followed by the mesodiencephalon, striatum, and cerebellum. This suggests that Cys is released from a neuronal compartment and might be involved in neurotransmission.     

Middle-ear development

Laser interferometry was used to measure umbo velocity in the developing BALB/c mouse middle ear at 133 pure-tone frequencies between 2.0 kHz and 40.0 kHz, all at a constant 100 dB sound pressure level. Umbo velocities increased with age across the entire frequency range, and reached adult-like levels by about 19 days between 2.0 and 22.0 kHz. Velocities at 28.0 and 34.0 kHz took 27 and 52 days respectively to reach adult-like levels.A simple middle-ear model utilizing compliance, resistance, and inertia elements matched the general trends of our velocity results and provided an indication of the anatomical basis for the growth in umbo velocity. The model suggested that velocity development at the lowest frequencies may be attributed to increases in tympanic membrane compliance. The model also indicated that both the frictional resistance of the middle ear and the inertia of the tympanic membrane and ossicles decreased during the growth period.At frequencies below 20.0 kHz, age-related increases in umbo velocity coincided with improvements in Nj₁ thresholds recorded from the round window and evoked potential thresholds obtained from the cochlear nucleus. These results indicated that the functional development of the middle-ear plays a major role in the development of hearing in the mouse.Portions of this work were presented at the Fifteenth Meeting of the Association for Research in Otolaryngology     

New Isopropyl Chromone and Flavanone Glucoside Compounds from the Leaves of Syzygium cerasiforme (Blume) Merr. & L.M.Perry and Their Inhibition of Nitric Oxide Production

A new isopropyl chromone ( 1 ) and a new flavanone glucoside ( 2 ) together with eleven known compounds ( 3–13 ) were isolated from the leaves of Syzygium cerasiforme (Blume) Merr. & L.M.Perry. Their structures were elucidated as 5,7-dihydroxy-2-isopropyl-6,8-dimethyl-4H-chromen-4-one ( 1 ), 5,7-dihydroxyflavanone 7-O-β-D-(6′′-O-galloylglucopyranoside) ( 2 ), strobopinin ( 3 ), demethoxymatteucinol ( 4 ), pinocembrin-7-O-β-D-glucopyranoside ( 5 ), (2S)-hydroxynaringenin-7-O-β-D-glucopyranoside ( 6 ), afzelin ( 7 ), quercetin ( 8 ), kaplanin ( 9 ), endoperoxide G3 ( 10 ), grasshopper ( 11 ), vomifoliol ( 12 ), litseagermacrane ( 13 ) by the analysis of HR-ESI-MS, NMR, and CD spectral data. Compounds 1 , 2 , 5 , 6 and 10 inhibited NO production on LPS-activated RAW264.7 cells with IC₅₀ values of 12.28±1.15, 8.52±1.62, 7.68±0.87, 9.67±0.57, and 6.69±0.34 μM, respectively, while the IC₅₀ values of the other compounds ranging from 33.38±0.78 to 86.51±2.98 μM, compared to that of the positive control, N^G-monomethyl-L-arginine acetate (L-NMMA) with an IC₅₀ value of 32.50±1.00 μM.     

Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.