Phencyclidine-like behavioral effects in pigeons induced by systemic administration of the excitatory amino acid antagonist, 2-amino-5-phosphonovalerate

A selective N-methyl-D-aspartate antagonist, DL-2-amino-5-phosphonovalerate, was found to produce PCP-like catalepsy, discriminative stimulus effects, and stereotyped operant responding in pigeons when administered intramuscularly. These results support the hypothesis that the behavioral effects of PCP-like drugs result at least in part from reduced neurotransmission at excitatory amino acid synapses utilizing N-methyl-D-aspartate preferring receptors.     

iRFP is a sensitive marker for cell number and tumor growth in high-throughput systems

GFP and luciferase are used extensively as markers both in vitro and in vivo although both have limitations. The utility of GFP fluorescence is restricted by high background signal and poor tissue penetrance. Luciferase throughput is limited in vitro by the requirement for cell lysis, while in vivo, luciferase readout is complicated by the need for substrate injection and the dependence on endogenous ATP. Here we show that near-infrared fluorescent protein in combination with widely available near-infrared scanners overcomes these obstacles and allows for the accurate determination of cell number in vitro and tumor growth in vivo in a high-throughput manner and at negligible per-well costs. This system represents a significant advance in tracking cell proliferation in tissue culture as well as in animals, with widespread applications in cell biology.     

A molecular dynamics simulation of bacteriophage T4 lysozyme.

An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented. The simulation was carried out with all hydrogen atoms modeled explicitly, the inclusion of all 152 crystallographic waters and at a temperature of 300 K. Temporal analysis of the trajectory versus energy, hydrogen bond stability, r.m.s. deviation from the starting crystal structure and radius of gyration, demonstrates that the simulation was both stable and representative of the average experimental structure. Average structural properties were calculated from the enzyme trajectory and compared with the crystal structure. The mean value of the C alpha displacements of the average simulated structure from the X-ray structure was 1.1 +/- 0.1 A; differences of the backbone phi and psi angles between the average simulated structure and the crystal structure were also examined. Thermal-B factors were calculated from the simulation for heavy and backbone atoms and both were in good agreement with experimental values. Relationships between protein secondary structure elements and internal motions were studied by examining the positional fluctuations of individual helix, sheet and turn structures. The structural integrity in the secondary structure units was preserved throughout the simulation; however, the A helix did show some unusually high atomic fluctuations. The largest backbone atom r.m.s. fluctuations were found in non-secondary structure regions; similar results were observed for r.m.s. fluctuations of non-secondary structure phi and psi angles. In general, the calculated values of r.m.s. fluctuations were quite small for the secondary structure elements. In contrast, surface loops and turns exhibited much larger values, being able to sample larger regions of conformational space. The C alpha difference distance matrix and super-positioning analyses comparing the X-ray structure with the average dynamics structure suggest that a 'hinge-bending' motion occurs between the N- and C-terminal domains.     

Nucleic acid constituent interactions

Hydrogen bonding contributes of the order of 5–15 kcal/mol base pair to the stability of the helix (electronic or intrinsic energy). This contribution is selective, i.e., there is a preferential stability of the Watson-Crick G-C pair relative to all other pairs. Stacking interactions contribute approximately of the same order as hydrogen bonding. Perhaps the most interesting aspect of the stacking interactions which emerges from the theoretical analysis is the fact that the stacking maxima are not necessarily at the angles the successive base pair plans assume in a regular double helix. Consequently some sequence dependent structure peculiarities may arise. That is, the double helix may have a fine structure contingent on the sequence of base pairs. Indeed such sequence dependent polymorphism has been reported in the recent literature and appears to influence the ability of aromatic drugs to intercalate into the helix. The solvent effect which is another factor of stability seems to decrease somewhat bonding scheme preferences. For example, in the model we used to estimate solvent effect, we find that the G-C pair formation is de-stabilized strongly in water, while the A-T pair formation is mildly enhanced. The continuum model of solvent effect leads to similar qualitative conclusions. Studies of backbone conformation indicate that only a limited range of conformational states are comparable with the helical configuration. Improved empirical methods are needed in order to successfully calculate backbone effects for relatively large segments of nucleic acids.     

Transmembrane AMPA receptor regulatory proteins and cornichon-2 allosterically regulate AMPA receptor antagonists and potentiators

AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [(3)H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [(3)H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [(3)H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [(3)H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.     

Molecular Dynamics Simulations Indicate that F87W,T185F-Cytochrome P450cam May Reductively Dehalogenate 1,1,1-Trichloroethane

Abstract

Cytochrome P450cam is capable of reductively dehalogenating several chlorinated alkanes at low, but measurable, rates. In previous investigations of structure-function relationships in this enzyme using molecular dynamics simulations, we noticed that 1,1,1-trichloroethane (TCA) exhibits a very high degree of mobility in the active site due to its smaller molecular volume relative to the native substrate, camphor(1,2). Several amino acid sidechains lining the active site also exhibit significant dynamic fluctuations, possibly as a result of poor steric complementarity to TCA. Guided by these results, we modeled double (F87W, T185F) and triple (F87W, T185F, V295I) mutants of P450cam, which provide additional bulk in the active site and increase the frequency of heme-substrate collision. Molecular dynamics simulations (300 ps on each protein) indicate that these mutants do not significantly perturb the three-dimensional fold of the enzyme, or local structure in the region of the active site. Both mutants bind the substrate more stably near the heme than the wild-type. Interestingly, however, the bulkier triple mutant seems to actually inhibit heme-substrate interactions relative to the double mutant. Over the final 200 ps of simulation, TCA is within 1 Å of nonbonded contact with the heme 25% more often in the double mutant versus the wild-type. The triple mutant, on the other hand, binds TCA within 1 Å of the heme only 15% as often as the wild-type. These results indicate that the double mutant may reductively dehalogenate TCA, a property not observed for the native protein. Implications for other experimentally measurable parameters are discussed.     

Molecular dynamics of subtilisin Carlsberg in aqueous and nonaqueous solutions

Crystal structures have recently appeared for the enzyme subtilisin Carlsberg in anhydrous acetonitrile and in water. To gain a mechanistic understanding of how the solvent environment affects protein structure and dynamics, we have performed molecular dynamics simulations on subtilisin Carlsberg in water and acetonitrile. We describe a 480 ps simulation of subtilisin in acetonitrile solution and a 450 ps simulation of subtilisin in water. Each simulation employed the all-atom AMBER force field. The calculated rms deviations, from their respective x-ray structures, were similar in each simulation, but ～0.5 ? higher in the acetonitrile simulation. Only in the acetonitrile simulation does one helix undergo a reversible partial unwinding, which lasted for about 100 ps. The other secondary structure elements remain intact or undergo modest fluctuations. In the aqueous simulation, the calculated and experimental temperature factors agree very well. In the acetonitrile simulation, however, the calculated temperature factors are much higher than the experimental values. The larger rms deviation and thermal fluctuations noted in the acetonitrile simulation are consistent with the requirement for protein cross-linking in this crystal and a recent two-dimensional NH-exchange nmr study on horse heart cytochrome c in nonaqueous solution. ? 1996 John Wiley & Sons, Inc.     

Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design

Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.     

Asymptotic Properties of Spearman’s Rank Correlation for Variables with Finite Support

The asymptotic variance and distribution of Spearman’s rank correlation have previously been known only under independence. For variables with finite support, the population version of Spearman’s rank correlation has been derived. Using this result, we show convergence to a normal distribution irrespectively of dependence, and derive the asymptotic variance. A small simulation study indicates that the asymptotic properties are of practical importance.     

Correlation of Tm,sequence, and ΔH of complementary RNA helices and comparison with DNA helices

T_m values of 16 fully complementary RNA duplexes with repeating base sequence have been employed as the empirical basis for developing a reliable and practical method for computing apparent enthalpies (ΔH _calc) for their helix → coil transitions. The approach taken is the same as in the accompanying investigation of DNA duplexes, although some of the computational variables of the “best-fit” function are necessarily different due to the distinguishing structural properties of the RNA-type helix. An excellent linear correlation was thus obtained between experimental T_m and ΔH _calc values. An equally good fit was obtained between T_m and ΔH _calc for five unrelated (to the 16 RNAs) decaribonucleotide duplexes. The differences in computational variables between the best-fit methods for RNA and DNA duplexes are shown to be a reflection of differences in cation binding and the effective local dielectric. The greater T_m dependence on G·C content of RNA helices than of DNA helices is shown to be due to a greater latitude of stacking stabilities of complementary dinucleotide fragments containing A·T than A·U base pairs.