Warming alters food web-driven changes in the CO2 flux of experimental pond ecosystems.

Evidence shows the important role biota play in the carbon cycle, and strategic management of plant and animal populations could enhance CO2 uptake in aquatic ecosystems. However, it is currently unknown how management-driven changes to community structure may interact with climate warming and other anthropogenic perturbations to alter CO2 fluxes. Here we showed that under ambient water temperatures, predators (three-spined stickleback) and nutrient enrichment synergistically increased primary producer biomass, resulting in increased CO2 uptake by mesocosms in early dawn. However, a 3°C increase in water temperatures counteracted positive effects of predators and nutrients, leading to reduced primary producer biomass and a switch from CO2 influx to efflux. This confounding effect of temperature demonstrates that climate scenarios must be accounted for when undertaking ecosystem management actions to increase biosequestration.

The contribution of cerebral vascular semicarbazide-sensitive amine oxidase to cerebral amyloid angiopathy in Alzheimer's disease.

Semicarbazide-sensitive amine oxidase (SSAO) catalyses the oxidative deamination of a variety of endogenous substrates, such as methylamine and aminoacetone, to produce highly reactive aldehydes, which are capable of inducing protein cross-linkage, beta amyloid (Abeta) aggregation and advanced glycation end-product formation. In the brain, SSAO is exclusively located on the blood vessels. Deposits of Abeta, the hallmark of Alzheimer's disease (AD), are closely associated with cerebral blood vessels, that is, cerebral amyloid angiopathy (CAA). In the present study, we examined whether SSAO-mediated deamination contributes to CAA in AD. We employed immunohistochemistry to examine the colocalization of SSAO and Abeta in post mortem brains of AD patients. To assess the role of SSAO-mediated deamination in the deposition of Abeta on blood vessel walls, we developed an in vitro blood vessel model using sections of human umbilical cord. We found a strong expression of SSAO colocalized with Abeta deposits on the blood vessels in AD brains. We also demonstrated that SSAO-mediated deamination increases the deposition of Abeta onto blood vessel walls. Our results support the hypothesis that cerebral vascular SSAO-catalysed deamination contributes to CAA in AD brains.

RNA backbone rotamers--finding your way in seven dimensions.

Despite the importance of local structural detail for a mechanistic understanding of RNA catalysis and binding functions, RNA backbone conformation has been recalcitrant to analysis. There are too many variable dihedral angles per residue, and their raw empirical distributions are poorly clustered. This study applies quality-filtering techniques (using resolution, crystallographic B factor and all-atom-steric clashes) to the backbone dihedral angle distributions from a selected 8636 residue RNA database. With noise levels significantly decreased, clear signal appears for the underlying angle preferences. We analyse the multidimensional backbone dihedral distributions within sugar-to-sugar 'suites' rather than chemical residues due to the greater base interaction and steric interdependence within the suite. The final result is a small library of RNA backbone rotamers, each represented by a data cluster in seven-dimensional dihedral space, which should provide valid conformations for nearly all RNA backbones encountered in experimental structures. We are in the process of improving that library, and developing tools and applications for it in structure determination and analysis.

Recovery of hippocampal cell proliferation and BDNF levels, both of which are reduced by repeated restraint stress, is accelerated by chronic venlafaxine.

The present study investigated the poststress (PS) cellular and molecular changes in the hippocampus of rats subjected to repeated restraint stress (RS) and the effects of chronic administration of an antidepressant drug, venlafaxine, on these changes. It was found that RS suppressed hippocampal cell proliferation, decreased brain-derived neurotrophic factor (BDNF) levels, and increased both the levels of copper/zinc superoxide dismutase (Cu/Zn-SOD) and the number of Cu/Zn-SOD immunostained hippocampal interneurons. In venlafaxine-treated rats, the changes in cell proliferation, BDNF levels, and the number of Cu/Zn-SOD interneurons returned to control levels on PS Days 21, 14, 7, respectively. In vehicle-injected rats, BDNF and the number of Cu/Zn-SOD interneurons returned to control levels on PS Days 21 and 14, respectively, but cell proliferation was still suppressed on PS Day 21. The stress-induced elevation of Cu/Zn-SOD protein remained during the 3-week PS period, and it was further increased by about 20% after 3 weeks of venlafaxine administration.

HeartMate III: pump design for a centrifugal LVAD with a magnetically levitated rotor.

A long-term, compact left ventricular assist device (LVAD), the HeartMate III, has been designed and fabricated, featuring a centrifugal pump with a magnetically levitated rotor. The pump has been optimized by in vitro testing to achieve a design point of 7 L/min against 135 mm Hg at high hydrodynamic efficiency (30%) and to be capable of up to 10 L/min under such a load. Furthermore, the pump has demonstrated no mechanical failures, low hemolysis (4-10 mg/dl plasma free Hb), and low thrombogenicity during six (40, 27, 59, 42, 27, and 49-day) in vivo bovine studies.

The Heartmate III: design and in vivo studies of a maglev centrifugal left ventricular assist device.

A compact implantable centrifugal left ventricular assist device (LVAD) (HeartMate III) featuring a magnetically levitated impeller is under development. The goal of our ongoing work is to demonstrate feasibility, low hemolysis, and low thrombogenicity of the titanium pump in chronic bovine in vivo studies. The LVAD is based on so-called bearingless motor technology and combines pump rotor, drive, and magnetic bearing functions in a single unit. The impeller is rotated (theta z) and levitated with both active (X, Y) and passive (Z, theta x, theta y) suspension. Six prototype systems have been built featuring an implantable titanium pump (69 mm diameter, 30 mm height) with textured blood contacting surfaces and extracorporeal electronics. The pumps were implanted in 9 calves (< or = 100 kg at implant) that were anticoagulated with Coumadin (2.5 < or = INR < or = 4.0) throughout the studies. Six studies were electively terminated (at 27-61 days), 1 study was terminated after the development of severe pneumonia and lung atelectasis (at 27 days) another study was terminated after cardiac arrest (at 2 days) while a final study is ongoing (at approximately 100 days). Mean pump flows ranged from 2 to 7 L/min, except for brief periods of exercise at 6 to 9 L/min. Plasma free hemoglobin ranged from 4 to 10 mg/dl. All measured biochemical indicators of end organ function remained within normal range. The pumps have met performance requirements in all 9 implants with acceptable hemolysis and no mechanical failures.

Protein design and folding: template trapping of self-assembled helical bundles.

An experimental system is described, permitting a detailed and systematic analysis of the factors governing self-assembly of amphipathic helices, e.g. to a four-helical bundle, a subject of major relevance for tertiary structure formation, protein folding and design. Following the Template Assembled Synthetic Proteins (TASP) approach, helices of different packing potential are competitively assembled in solution with a preformed two-helix TASP molecule, and after equilibration are covalently attached ('template trapping') via chemoselective thioether formation. The quantitative analysis of the individual TASP molecules by high performance liquid chromatography (HPLC) and electrospray mass spectrometry (ES-MS) allows the delineation of the role of complementary packing in helix bundle formation. The procedure established represents a general tool for the experimental verification of modern concepts in molecular recognition.

SymROP: ROP protein with identical helices redesigned by all-atom contact analysis and molecular dynamics.

Experience has shown that protein redesigns (using the backbone from a known protein structure) are far more likely to produce well-ordered, native-like structures than are true de novo designs. Therefore, to design a four-helix bundle made of identical short helices, we here proceed by an extensive redesign of the ROP protein. A fully symmetrical SymROP sequence derived from ROP was chosen by modeling ideal-geometry side chains, including hydrogens, while maintaining the "goodness-of-fit" of side-chain packing by calculating all-atom contact surfaces with the Reduce and Probe programs. To estimate the probable extent of backbone movement and side-chain mobility, restrained molecular dynamics simulations were compared for candidate sequences and controls, including substitution of Abu for all or half the core Ala residues. The resulting 17-residue designed sequence is 41% identical to the relevant regions in ROP. SymROP is intended for construction by the Template Assembled Synthetic Proteins approach, to control the bundle topology, to use short helices, and to allow blocked termini and unnatural amino acids. ROP protein has been a valuable system for studying helical protein structure because of its simplicity and regularity within a structure large enough to have a real hydrophobic core. The SymROP design carries that simplicity and regularity even further.

The penultimate rotamer library.

All published rotamer libraries contain some rotamers that exhibit impossible internal atomic overlaps if built in ideal geometry with all hydrogen atoms. Removal of uncertain residues (mainly those with B-factors >/=40 or van der Waals overlaps >/=0.4 A) greatly improves the clustering of rotamer populations. Asn, Gln, or His side chains additionally benefit from flipping of their planar terminal groups when required by atomic overlaps or H-bonding. Sensitivity to skew and to the boundaries of chi angle bins is avoided by using modes rather than traditional mean values. Rotamer definitions are listed both as the modal values and in a preferred version that maximizes common atoms between related rotamers. The resulting library shows significant differences from previous ones, differences validated by considering the likelihood of systematic misfitting of models to electron density maps and by plotting changes in rotamer frequency with B-factor. Few rotamers now show atomic overlaps in ideal geometry; those overlaps are relatively small and can be understood in terms of bond angle distortions compensated by favorable interactions. The new library covers 94.5% of examples in the highest quality protein data with 153 rotamers and can make a significant contribution to improving the accuracy of new structures. Proteins 2000;40:389-408.

Caregiver distress. Related to disruptive behaviors on special care units versus traditional long-term care units.

The link between staff stress and exposure to disruptive behaviors is an important issue in long-term care settings. This study compared the perceptions of two groups of formal caregivers (staff) regarding their distress from the behaviors of residents in their care. Staff on special care units for dementia were less distressed with disruptive behaviors than comparable staff on traditional units, although they reported higher exposure to these behaviors. These results were related to different perceptions of intent to harm and expectations of physical aggression as "part of the job." Implications for nursing include education and support for staff to enhance the quality of life for residents and staff on units where disruptive behaviors occur.

Neuroprotective agents.

Although basic research has revealed many mechanisms involved in the repair or elimination of damaged neurons, turning these mechanisms into clinically useful neuroprotective interventions is a slow process. Numerous neurotrophic factors seem to mediate neuronal repair and viability, but because the neurotrophic factors are proteins or polypeptides, they cannot be given orally and do not enter the brain if given intravenously. Tapping into the neuroprotective potential of the neurotrophic factor mechanisms must await further developments. Similarly, pharmacological agents that protect damaged neurons by reducing glutamate excitotoxicity, by scavenging free radicals, or by increasing adenosine inhibitory influences, are not ready yet for widespread clinical use. Also, appropriate therapeutic protocols for currently available neuroprotective agents such as vitamin E, selegiline, and NSAIDs remain to be determined. Given the rate of advance of research in this area, however, meaningful neuroprotection and neurorescue will be attainable in the very near future. In the meantime, neuron damaging oxidative stress can be kept in check by insuring adequate dietary sources of antioxidants. Although there is as yet little or no scientific evidence that dietary antioxidants are neuroprotective, the consumption of high antioxidant foods, such as blueberries and strawberries, is appealing to most people regardless of any neuroprotective potential.

Visualizing and quantifying molecular goodness-of-fit: small-probe contact dots with explicit hydrogen atoms.

The technique of small-probe contact dot surfaces is described as a method for calculating and displaying the detailed atomic contacts inside or between molecules. It allows one both to measure and to visualize directly the goodness-of-fit of packing interactions. It requires both highly accurate structures and also the explicit inclusion of all hydrogen atoms and their van der Waals interactions. A reference dataset of 100 protein structures was chosen on the basis of resolution (1.7 A or better), crystallographic R-value, non-homology, and the absence of any unusual problems. Hydrogen atoms were added in standard geometry and, where needed, with rotational optimization of OH, SH, and NH+3 positions. Side-chain amide orientations were corrected where required by NH van der Waals clashes, as described in the accompanying paper. It was determined that, in general, methyl groups pack well in the default staggered conformation, except for the terminal methyl groups of methionine residues, which required rotational optimization. The distribution of serious clashes (i.e. non-H-bond overlap of >/=0.4 A) was studied as a function of resolution, alternate conformations, and temperature factor (B), leading to the decision that packing and other structural features would not be analyzed for residues in 'b' alternate conformations or with B-factors of 40 or above. At the level of the fine details analyzed here, structural accuracy improves quite significantly over the range from 1.7 to 1.0 A resolution. These high-resolution structures show impressively well-fitted packing interactions, with some regions thoroughly interdigitated and other regions somewhat sparser. Lower-resolution structures or model structures could undoubtedly be improved in accuracy by the incorporation of this additional information: for example, nucleic acid structures in non-canonical conformations are often very accurate for the bases and much less reliable for the backbone, whose conformation could be specified better by including explicit H atom geometry and contacts. The contact dots are an extremely sensitive method of finding problem areas, and often they can suggest how to make improvements. They can also provide explanations for structural features that have been described only as empirical regularities, which is illustrated by showing that the commonest rotamer of methionine (a left-handed spiral, with all chi values near -60 degrees) is preferred because it provides up to five good H atom van der Waals contacts. This methodology is thus applicable in two different ways: (1) for finding and correcting errors in structure models (either experimental or theoretical); and (2) for analyzing interaction patterns in the molecules themselves.

Asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation.

Small-probe contact dot surface analysis, with all explicit hydrogen atoms added and their van der Waals contacts included, was used to choose between the two possible orientations for each of 1554 asparagine (Asn) and glutamine (Gln) side-chain amide groups in a dataset of 100 unrelated, high-quality protein crystal structures at 0.9 to 1.7 A resolution. For the movable-H groups, each connected, closed set of local H-bonds was optimized for both H-bonds and van der Waals overlaps. In addition to the Asn/Gln "flips", this process included rotation of OH, SH, NH3+, and methionine methyl H atoms, flip and protonation state of histidine rings, interaction with bound ligands, and a simple model of water interactions. However, except for switching N and O identity for amide flips (or N and C identity for His flips), no non-H atoms were shifted. Even in these very high-quality structures, about 20 % of the Asn/Gln side-chains required a 180 degrees flip to optimize H-bonding and/or to avoid NH2 clashes with neighboring atoms (incorporating a conservative score penalty which, for marginal cases, favors the assignment in the original coordinate file). The programs Reduce, Probe, and Mage provide not only a suggested amide orientation, but also a numerical score comparison, a categorization of the marginal cases, and a direct visualization of all relevant interactions in both orientations. Visual examination allowed confirmation of the raw score assignment for about 40 % of those Asn/Gln flips placed within the "marginal" penalty range by the automated algorithm, while uncovering only a small number of cases whose automated assignment was incorrect because of special circumstances not yet handled by the algorithm. It seems that the H-bond and the atomic-clash criteria independently look at the same structural realities: when both criteria gave a clear answer they agreed every time. But consideration of van der Waals clashes settled many additional cases for which H-bonding was either absent or approximately equivalent for the two main alternatives. With this extra information, 86 % of all side-chain amide groups could be oriented quite unambiguously. In the absence of further experimental data, it would probably be inappropriate to assign many more than this. Some of the remaining 14 % are ambiguous because of coordinate error or inadequacy of the theoretical model, but the great majority of ambiguous cases probably occur as a dynamic mix of both flip states in the actual protein molecule. The software and the 100 coordinate files with all H atoms added and optimized and with amide flips corrected are publicly available.

Asparagine and glutamine rotamers: B-factor cutoff and correction of amide flips yield distinct clustering.

Previous rotamer libraries showed little significant clustering for asparagine chi2 or glutamine chi3 values, but none of those studies corrected amide orientations or omitted disordered side chains. The current survey used 240 proteins at /=0.4 A). All H atoms were added and optimized, and amide orientation was flipped by 180 degrees if required by H bonding or atomic clashes. A side chain was included only if its amide orientation was clearly determined and if no atom had a B factor >/=40, alternate conformation, or severe clash; that selection process yielded 1,490 Asn and 863 Gln side chains. Clear clustering was observed for Asn chi2 and Gln chi3 (except when Gln chi2 is trans). For Gln, five major and four minor rotamers cover 87% of examples. For Asn, there are seven backbone-independent rotamers covering 94% of examples plus rotamers specified for strictly alpha-helical, beta, and left-handed (+phi) Asn. Although the strongest influence on chi angles is avoidance of atomic clashes (especially with the NH2 hydrogens), some Asn or Gln rotamers are influenced by favorable van der Waals contacts and others by specific local H-bond patterns.

Genetic markers on HIV-1 gp120 C2-V3 region associated with the expression or absence of cognitive motor complex in HIV/AIDS.

In the CNS, HIV-1 causes cognitive motor complex (CMC) in about 30-40% of patients. To explain CMC physiopathology: disequilibrium of cytokine networks, calcium influx, free radicals and toxic effects by HIV-1 have been evoked. Neurotropic mutants have not been unambiguously proven nor 'variants' of HIV-1 with biological properties that could cause CMC. By computerized analysis of gp120 C2-V3 subtype B sequences from retroviral databases, and applying stringent criteria, we found: (i) mutations specific for CMC; (ii) mutations associated with the absence of CMC (N-CMC); (iii) mutations with specificity for the geographical region of origin, and finally (iv) shared mutations representing 'hot spots.' We suggest that the capability to cause or not to cause CMC may be present in the virus prior to infection. In the future, these markers could be used to guide treatments with novel neuroprotective regimes.

Folding kinetics of a fluorescent variant of monomeric lambda repressor.

A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 micro(s) in the absence of denaturants. All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.

Antiamyloid strategies for the treatment of Alzheimer's disease.

Although the specific causes of Alzheimer's disease have not yet been determined, considerable circumstantial evidence implicates beta-amyloid, an insoluble polypeptide made up of 39 to 42 amino acids, in the continuing destruction of brain cells that results in the progressive deterioration of the patient's mental ability. The toxic actions of beta-amyloid appear to be due to free radicals generated by a portion of the beta-amyloid molecule. These free radicals damage various parts of the neuron and lead to increased intracellular calcium which is also toxic. beta-Amyloid is formed by the aberrant processing of a much larger precursor protein that is made when cells are damaged. The normal processing of this precursor protein not only prevents the formation of beta-amyloid, but produces a soluble protein that regulates the entry of calcium into neurons and has cytoprotective actions. Interventions to prevent the destruction of neurons and the disruption of brain function by beta-amyloid include the administration of antioxidants and free radical scavengers to reduce further neural damage from deposits of beta-amyloid, the activation of various growth factors to repair damaged cells and restore their functions, and the stimulation of the normal processing of the precursor protein not only to aid in neural repair but more importantly to prevent the formation of additional beta-amyloid.