Intranasal dexmedetomidine in healthy beagles: An echocardiographic and pharmacokinetic/pharmacodynamic study.

The aim of this study was to compare the sedative and cardiovascular effects of dexmedetomidine (DEX) administrated via intranasal (IN) and intramuscular (IM) routes. This masked, randomised, crossover study used six male beagle dogs, receiving 0.02 mg/kg dexmedetomidine either IN (DEX-IN) or IM (DEX-IM), and an equal volume of saline by the alternative route. Dexmedetomidine plasma concentration was measured before (TB) and at time points (T) 2, 5, 10, 15, 30, 45, 60, 90 and 120 min after drug administration (T0). Physiological variables, sedation scores and sedation times were recorded until recovery. Echocardiography was performed at TB and between T20-T40. Repeated data over time were analysed using a Scheirer-Ray-Hare test. Other data were compared using a Wilcoxon or Student's t test. Times from T0 to sternal position and from lateral to sternal position were longer for DEX-IN than DEX-IM (P = 0.018 and P = 0.009, respectively). Time from sternal to standing position was shorter for DEX-IN than DEX-IM (P = 0.03). Dexmedetomidine plasma concentrations were significantly lower for DEX-IN than DEX-IM from T10 to T120. Heart rate was significantly lower for DEX-IM than DEX-IN from T5 to T20. Echocardiography showed a decrease in systolic function and calculated cardiac output in DEX-IM as compared to baseline. The DEX concentration-sedation curve for DEX-IN as compared to DEX-IM was leftward shifted, whereas the IN and IM DEX concentration-variation-in-heart-rate curves overlapped. Although reaching lower plasma concentrations, IN dexmedetomidine produced similar sedation to IM dexmedetomidine without affecting cardiovascular function.

Ignition's glow: Ultra-fast spread of global cortical activity accompanying local "ignitions" in visual cortex during conscious visual perception.

Despite extensive research, the spatiotemporal span of neuronal activations associated with the emergence of a conscious percept is still debated. The debate can be formulated in the context of local vs. global models, emphasizing local activity in visual cortex vs. a global fronto-parietal "workspace" as the key mechanisms of conscious visual perception. These alternative models lead to differential predictions with regard to the precise magnitude, timing and anatomical spread of neuronal activity during conscious perception. Here we aimed to test a specific aspect of these predictions in which local and global models appear to differ - namely the extent to which fronto-parietal regions modulate their activity during task performance under similar perceptual states. So far the main experimental results relevant to this debate have been obtained from non-invasive methods and led to conflicting interpretations. Here we examined these alternative predictions through large-scale intracranial measurements (Electrocorticogram - ECoG) in 43 patients and 4445 recording sites. Both ERP and broadband high frequency (50-150 Hz - BHF) responses were examined through the entire cortex during a simple 1-back visual recognition memory task. Our results reveal short latency intense visual responses, localized first in early visual cortex followed (at ∼200 ms) by higher order visual areas, but failed to show significant delayed (300 ms) visual activations. By contrast, oddball image repeat events, linked to overt motor responses, were associated with a significant increase in a delayed (300 ms) peak of BHF power in fronto-parietal cortex. Comparing BHF responses with ERP revealed an additional peak in the ERP response - having a similar latency to the well-studied P3 scalp EEG response. Posterior and temporal regions demonstrated robust visual category selectivity. An unexpected observation was that high-order visual cortex responses were essentially concurrent (at ∼200 ms) with an ultra-fast spread of signals of lower magnitude that invaded selected sites throughout fronto-parietal cortical areas. Our results are compatible with local models in demonstrating a clear task-dependence of the 300 ms fronto-parietal activation. However, they also reveal a more global component of low-magnitude and poor content selectivity that rapidly spreads into fronto-parietal sites. The precise functional role of this global "glow" remains to be elucidated.

Objective pain assessment after ureteral reimplantation: comparison of open versus robotic approach.

INTRODUCTION: While open ureteral reimplantation is the gold standard of surgical intervention for vesicoureteral reflux (VUR), minimally invasive approaches offer the potential benefits of decreased postoperative pain, improved cosmesis, and shorter hospital stay and convalescence. Studies comparing open and minimally invasive surgery with respect to postoperative pain in children have been inconclusive. OBJECTIVE: We sought to compare postoperative pain in children undergoing open versus robotic ureteral reimplantation by using age-appropriate, validated pain assessment scales. METHODS: A prospective cohort of all patients enrolled in an Institutional Review Board-approved VUR surgery registry between July 2010 and February 2013 was analyzed. Patients who underwent endoscopic treatment or who received caudal or epidural anesthesia were excluded. Age-appropriate, validated pain scales ranging from 0 to 10 were utilized for pain assessment. Pain scores and narcotic doses administered on the first postoperative day were analyzed. RESULTS: Of the 34 subjects included, 11 underwent open intravesical reimplantation, while 23 patients underwent robotic extravesical reimplantation. Table 1 displays patient characteristics and results of pain assessment. Robotic surgery was associated with lower narcotic requirement compared to open surgery (P < 0.05). The difference in pain scores between the two cohorts approached, but did not reach, statistical significance (P = 0.12). However, the percentage of patients with mild or no pain (57% robotic, 27% open) versus severe pain (9% robotic, 45% open) was notably different between the two cohorts. DISCUSSION: Previous studies addressing the effect of surgical modality on pediatric postoperative pain are limited by their reliance on narcotic administration as an indirect surrogate for measuring pain. In the present study, postoperative pain was assessed with narcotic requirements and consistently collected validated pain scores, which more accurately reflect a patient's perceived pain. Although there was no significant difference in subjective pain scores between patients undergoing open versus robotic reimplantation, the percentage of patients with mild or no pain (57% robotic, 27% open) versus severe pain (9% robotic, 45% open) was notably different between the two cohorts. This study was limited by a lack of randomization as well as small sample size, which did not allow for age sub-group analysis or small differences to be statistically significant. CONCLUSIONS: In the present study, robotic ureteral reimplantation was associated with lower narcotic requirement compared to open surgery, and lower intensity of postoperative pain according to a direct pain assessment tool. Larger sample sizes are necessary to strengthen statistical comparisons.

Normal form from biological motion despite impaired ventral stream function.

We explored the extent to which biological motion perception depends on ventral stream integration by studying LG, an unusual case of developmental visual agnosia. LG has significant ventral stream processing deficits but no discernable structural cortical abnormality. LG's intermediate visual areas and object-sensitive regions exhibit abnormal activation during visual object perception, in contrast to area V5/MT+ which responds normally to visual motion (Gilaie-Dotan, Perry, Bonneh, Malach, & Bentin, 2009). Here, in three studies we used point light displays, which require visual integration, in adaptive threshold experiments to examine LG's ability to detect form from biological and non-biological motion cues. LG's ability to detect and discriminate form from biological motion was similar to healthy controls. In contrast, he was significantly deficient in processing form from non-biological motion. Thus, LG can rely on biological motion cues to perceive human forms, but is considerably impaired in extracting form from non-biological motion. Finally, we found that while LG viewed biological motion, activity in a network of brain regions associated with processing biological motion was functionally correlated with his V5/MT+ activity, indicating that normal inputs from V5/MT+ might suffice to activate his action perception system. These results indicate that processing of biologically moving form can dissociate from other form processing in the ventral pathway. Furthermore, the present results indicate that integrative ventral stream processing is necessary for uncompromised processing of non-biological form from motion.

Antibodies against the VRT101 laminin epitope correlate with human SLE disease activity and can be removed by extracorporeal immunoadsorption.

OBJECTIVE: We have previously shown that murine pathogenic lupus autoantibodies bind to VRT101, a 21-mer peptide located at the globular part of the laminin-alpha chain. In this study, we evaluated whether VRT101 also serves as a target for human lupus antibodies, upholding the hypothesis that VRT101 may serve as a potential target in the therapy of lupus. METHODS: Anti-VRT101 and anti-dsDNA reactivity were measured in the serum of lupus patients and compared with that of healthy individuals and patients with other rheumatic disorders. Statistical correlations between disease activity measured by the SLEDAI-2k scale and compatible serum anti-VRT101 and anti-dsDNA levels were defined. A VRT101-coupled sepharose column was assessed for its efficacy in removing serum anti-VRT101 antibody and its safety in extracorporeal apheresis in sheep. RESULTS: Anti-VRT101 and anti-dsDNA antibodies were significantly higher in SLE patients compared with patients with other rheumatic conditions. A high degree of correlation was detected between anti-VRT101 levels and the SLEDAI-2k activity in patients with SLE. Immunoadsorption of lupus patients' sera on the VRT101-sepharose column removed most of the anti-VRT101 antibodies. The column was found to transfer effectively 3l of normal sheep plasma without significant removal of non-specific antibodies or other proteins. CONCLUSIONS: Anti-VRT101 anibodies are abundantly detected in the serum of patients with SLE and correlate with disease activity. Specific removal of serum anti-VRT101 by extracorporeal plasmapheresis with specific immunoadsorption on the VRT101-coupled sepharose columns may serve as a new therapeutic tool for specific immunoadsorption of pathogenic antibodies in SLE patients.

Application of a static fluorescence-based cytometer: the CellScan in clinical immunology.

The CellScan system is a laser scanning cytometer which enables repetitive fluorescence intensity (FI) and polarization (FP) measurements in living cells, as a means of monitoring lymphocyte activation. By monitoring FP changes in peripheral blood lymphocytes (PBL) following exposure to antigenic stimuli, the CellScan may have a role in the diagnosis of autoimmune diseases. Monitoring changes in FI and FP in PBLs from patients with atherosclerosis following exposure to various stimuli, has illustrated the role of the immune system in the atherosclerotic process. The CellScan has also been evaluated as a diagnostic tool for drug-induced allergy, based on FP reduction in PBLs following incubation with the suspected drugs. FI and FP changes in cancer cells have been found to correlate with the cytotoxic effect of different anti-neoplastic drugs, illustrating the potential role of the CellScan system in clinical oncology. In conclusion, the CellScan is a promising new tool with a variety of applications in cell biology, immunology, cancer research and clinical pharmacology.

Selective fovea-related deprived activation in retinotopic and high-order visual cortex of human amblyopes.

Amblyopia is a visual disorder starting at early childhood and characterized by reduced visual acuity not of optical origin or due to any eye disease. One expression of such an anomalous early visual experience is abnormal foveal vision. In a previous fMRI study, faces that were presented to amblyopic eyes evoked little response compared to houses in high-order visual areas. Patients also demonstrated reduced recognition of facial expression, raising the possibility that these face-selective abnormalities are related to foveal vision deficit. Whether this deficit originates in low-level processing or is mediated by compromised activation in high-order visual areas is unresolved. In the present functional magnetic resonance imaging (fMRI) study, we explored the impact of amblyopia on the representation of object images presented in foveally biased central versus peripheral retinotopic eccentricities through manipulation of object size. Small and large pictures were correlated to visual acuities of 6/6 and 6/60, respectively. In low-level visual areas, the amblyopic eye showed significantly reduced activation for centrally placed, small pictures than the sound eye, while activation to large pictures was only slightly reduced. Similarly, in high-order visual areas, the amblyopic eye showed marked reduction in activation in the fusiform gyrus, with normal activation in the collateral sulcus. The center/periphery size-related amblyopic outcomes of this study support a "bottom-up" nature of the center-periphery effect observed in high-order visual areas. Taken together, these findings point to the regional extent and functional selectivity of fovea-related cortical reorganization that is related to abnormal visual development of one eye.

Synthesis and biological activity of the four stereoisomers of 4-methyl-3-heptanol: main component of the aggregation pheromone of Scolytus amygdali.

Stereoisomers of 4-methyl-3-heptanol are major components of aggregation pheromones of bark beetles and trail pheromones of ants. Recently, (3S,4S)-4-methyl-3-heptanol (I) has been tentatively identified as the main component of the aggregation pheromone of the almond bark beetle, Scolytus amygdali (Coleoptera: Scolytidae). The four stereoisomers of 4-methyl-3-heptanol were prepared and bioassayed. Key steps included preparation of chiral 4-methyl-3-heptanones using SAMP and RAMP reagents, reduction to the corresponding alcohols, and stereospecific transesterification with vinyl acetate with lipase AK catalysis. In field tests, only (3S,4S)-4-methyl-3-heptanol attracted beetles in combination with the synergist (3S,4S)-4-methyl-3-hexanol, whereas (3R,4S)- and (3R,4R)-4-methyl-3-heptanols were inhibitory.

Sex pheromone of the citrus mealybug Planococcus citri: synthesis and optimization of trap parameters.

A simple synthesis of the pheromone of the citrus mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae), has been developed. Various factors affecting capture of males have been assessed to optimize the trap design and to develop a lure with high efficacy and longevity. Male capture was the same with the racemic and chiral pheromone; technical pheromone (85% purity) was statistically as attractive as pure pheromone (97%). A special formulation was used to determine the actual release rate of the pheromone under field conditions as related to male capture. Generally, plate traps caught more males than delta traps, and large traps caught more than small ones. The effects of aging on the performance of three types of rubber dispensers were evaluated. It was found that the American dispenser displayed the most consistent trapping performance and could be used for monitoring for at least 16 wk with a load of 200 microg of pheromone. The dose-response of the males to sex pheromone was tested within the range of 25-1,600 microg.

Area-specific amblyopic effects in human occipitotemporal object representations.

The role of early visual experience in the establishment of human high-order visual areas is poorly understood. Here we investigated this issue using human amblyopia--a developmental visual disorder, which manifests a central vision (acuity) deficit. Previous fMRI studies of amblyopes have described abnormal functional activations in early retinotopic areas. Here we report the surprising finding of a selective object-related abnormality in high-order occipitotemporal cortex. Specifically, we found that face-related cortical areas show a severe disconnection from the amblyopic eye, while building-related regions remain essentially normal. The selectivity of the deficit highlights the differential computations performed in the different object-related areas and is compatible with the suggested association of face regions with analysis of fine detail.

Sex pheromone of the vine mealybug, Planococcus ficus in Israel: occurrence of a second component in a mass-reared population.

Two pheromonal components were detected in airborne collections from the vine mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) mass-reared on potato sprouts. The compounds were identified as (S)-lavandulyl senecioate (I) and (S)-lavandulyl isovalerate (II) by GC and GC-MS by comparison with synthetic standards. Chiral GC analysis on a cyclodextrin column established their chirality. Compound I was identified recently as the sex pheromone of P. ficus in California. The attraction of vine mealybug males to both components I and II was demonstrated in a Petri dish bioassay and in a flight assay in the rearing chamber. Indoors, both compounds displayed a similar level of attractiveness to the mass-reared males. However, trials in a vineyard indicated that feral males were attracted only to compound I. Reanalysis of the airborne pheromone indicated that laboratory first generation daughters of females that were collected in the vineyard produce only (S)-lavandulyl senecioate (I). The relative amount of (S)-lavandulyl isovalerate (II) increased gradually in each subsequent generation of P. ficus reared on potatoes. These findings indicate that feral P. ficus mealybugs produce and respond only to (S)-lavandulyl senecioate (I), whereas mealybugs that were reared in the laboratory on potato sprouts produce and respond to both (S)-lavandulyl senecioate (I) and (S)-lavandulyl isovalerate (II).

X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (-)-huperzine B: structural evidence for an active site rearrangement.

Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.

Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine.

Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (k(i) = 2.0 M(-1) min(-1)), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is >1600-fold higher (k(i) = 3300 M(-1) min(-1)). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (k(i) = 9 x 10(4) and 5 x 10(5) M(-1) min(-1), respectively). Spontaneous reactivation of all four conjugates is very slow, with <10% reactivation being observed for the Torpedo enzyme after 48 h. The crystal structure of the conjugate of rivastigmine with Torpedo acetylcholinesterase was determined to 2.2 A resolution. It revealed that the carbamyl moiety is covalently linked to the active-site serine, with the leaving group, (-)-S-3-[1-(dimethylamino)ethyl]phenol, being retained in the "anionic" site. A significant movement of the active-site histidine (H440) away from its normal hydrogen-bonded partner, E327, was observed, resulting in disruption of the catalytic triad. This movement may provide an explanation for the unusually slow kinetics of reactivation.

3D structure of Torpedo californica acetylcholinesterase complexed with huprine X at 2.1 A resolution: kinetic and molecular dynamic correlates.

Huprine X is a novel acetylcholinesterase (AChE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 A resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.

Quantitative GC analysis of secondary alcohol pheromones: determination of release rate of red palm weevil, Rhynchophorus ferrugineus, pheromone from lures.

Aliphatic secondary alcohols are components of several aggregation pheromones of important beetle and weevil pests. Some of these pheromones are used frequently for the monitoring and mass trapping of the relevant insects. We encountered severe difficulties in direct GC quantitative analysis of these compounds. Therefore, we developed a simple GC analysis of secondary alcohols convening them to trifluoroacetyl derivatives and using secondary alcohol acetates as internal standards. This method was applied for the quantitative analysis of several secondary alcohols, including the aggregation pheromone components of the almond bark beetle and the red palm weevil. The release rate of the latter pheromone from commercial lures was also determined.

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

Application of a gamma model of absorption to oral cyclosporin.

BACKGROUND: Some drugs, such as cyclosporin, exhibit flat and delayed absorption profiles, with a correlation between the delay and the peak width. Such profiles can be described by an absorption model in which the absorption rate is derived from a gamma distribution (of which the classical first-order absorption model is a special case). OBJECTIVE: To develop a model for the pharmacokinetics of extravascular administration of cyclosporin and apply it to a study of the pharmacokinetics of cyclosporin microemulsion in stable renal transplant recipients. PATIENTS AND PARTICIPANTS: 21 renal transplant patients receiving oral cyclosporin microemulsion 75 to 175 mg twice daily. METHODS: The equation of the plasma concentration-time curve after oral administration was expressed as a convolution product between the absorption rate and a multi-exponential impulse response. The convolution integral was computed analytically and expressed in terms of the incomplete gamma function. Cyclosporin was assayed by liquid chromatography/mass spectrophotometry. The model was fitted by nonlinear regression, using a specially developed program. RESULTS: The gamma model yielded a good fit in all of the 21 patients studied. Attempts to fit the same data by a classical exponential with lag-time model failed in most patients. CONCLUSIONS: This model could simplify the Bayesian monitoring of cyclosporin therapy.

A hierarchical axis of object processing stages in the human visual cortex.

How are objects represented in the human visual cortex? Two conflicting theories suggest either a holistic representation, in which objects are represented by a collection of object templates, or a part-based representation, in which objects are represented as collections of features or object parts. We studied this question using a gradual object-scrambling paradigm in which pictures of objects (faces and cars) were broken in a stepwise manner into an increasing number of blocks. Our results reveal a hierarchical axis oriented anterior--posteriorly in the organization of ventral object-areas. Along this axis, representations are arranged in bands of increasing sensitivity to image scrambling. The axis starts in early visual areas through retinotopic areas V4/V8 and continues into the lateral-occipital sulcus dorsally and the posterior fusiform girus ventrally, corresponding together to the previously described object-related lateral occipital complex (LOC). Regions showing the highest sensitivity to scrambling tended to be located at the most anterior-lateral regions of the complex. In these more anterior regions, breaking the images into 16 parts produced a significant reduction in activation. Interestingly, activation was not affected when images were cut in two halves, either horizontally or vertically. Car images generally produced a weaker activation compared to faces in the lateral occipital complex but showed the same tendency of increased scrambling sensitivity along the anterior--posterior axis. These results suggest the existence of a hierarchical axis along ventral occipito-temporal object-areas, in which the neuronal properties shift from sensitivity to local object features to a more global and holistic representation.

Modulation of arachidonate and docosahexaenoate in Morone chrysops larval tissues and the effect on growth and survival.

The extent to which extreme dietary levels of arachidonate (AA) and/or docosahexaenoate (DHA) modulate lipid composition in the body tissues and consequently affect growth and survival in freshwater Morone larvae species was examined. White bass, M. chrysops, larvae (day 24-46) were fed Artemia nauplii enriched with algal oils containing varying proportions of AA and DHA (from 0 to over 20% the total fatty acids). Growth was significantly reduced (P< 0.05) in larvae fed a DHA-deficient Artemia diet. Increases in dietary levels of AA also were associated with a significant growth reduction. However, the inhibitory effect of AA on larvae growth could be suppressed by the dietary addition of DHA (at a level of 21.6% of the total fatty acids in enrichment lipids). Larval brain + eyes tissue accumulated over 10 times more DHA than AA in its structural lipids (phosphatidylcholine, phosphatidylethanolamine) at any dietary ratio. In contrast, DHA accumulation, as compared to AA, in gill lipids declined considerably at higher than 10:1 DHA/AA tissue ratios. DHA and eicosapentaenoic acid (EPA) contents in brain + eyes tissue were most sensitive to competition from dietary AA, being displaced from the tissue at rates of 0.36 +/- 0.07 mg DHA and 0.46 +/- 0.11 mg EPA per mg increase in tissue AA, and 0.55 +/- 0.14 mg AA per mg increase in tissue DHA. On the other hand, AA and EPA levels in gill tissue were most sensitive to dietary changes in DHA levels; AA was displaced at rates of 0.37 +/- 0.11 mg, whereas EPA increased at rates of 0.68 +/- 0.28 mg per mg increase in tissue DHA. Results suggest that balanced dietary DHA/AA ratios (that allow DHA/AA ratios of 2.5:1 in brain + eyes tissue) promote a high larval growth rate, which also correlates with maximal regulatory response in tissue essential fatty acids.

Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II.

Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.