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Biomedical subjects

J S Richardson

Publications and source records attributed to J S Richardson.

At least 19 recordsLinked to original sources
PubMed2004

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.

Animals↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

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.

Animals↗
PubMed2000

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.

Amino Acids↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

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.

Crystallography, X-Ray↗
PubMed1999

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.

Algorithms↗
PubMed1999

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 </=1.7 A resolution with <50% homology and <30 clashes per thousand atoms (atomic overlap >/=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.

Asparagine↗
PubMed1999

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.

Adolescent↗
PubMed1999

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.

Central Nervous System Diseases↗
PubMed1998

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.

Amino Acid Substitution↗
PubMed1998

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.

Journal Article↗
PubMed1997

Autonomic dysreflexia: pharmacological management of hypertensive crises in spinal cord injured patients.

Interruption of autonomic pathways by spinal cord injury (SCI) causes dysfunctional autonomic dysreflexia (AD), which was first described in 1917, still remains unrecognized by those in the medical profession not involved in SCI care. Autonomic dysreflexia is a syndrome generally manifest by cardiovascular symptoms and characterized by paroxysmal hypertension. These symptoms appear in patients with spinal cord injury above the sympathetic outflow from the spinal cord (T6). Since patients with high level SCI are usually hypotensive, the high blood pressures that develop during AD represent pressure changes of a magnitude that can cause cerebrovascular accidents and death of the subject. We discuss the therapeutic interventions that abate and curtail the symptoms and prevent the catastrophic sequelae of autonomic dysreflexia.

Autonomic Nervous System Diseases↗
PubMed1996

Cholesterol protects PC12 cells from beta-amyloid induced calcium disordering and cytotoxicity.

THE neurotoxic action of beta-amyloid seems to play an important role in the pathogenesis of Alzheimer's disease. The disruption of calcium homeostasis by beta-amyloid has been suspected to be the mechanism of its neurotoxicity. We found that beta-amyloid 25-35 induces a rapid increase in cytosolic calcium of PC12 cells, and subsequently, a dramatic decrease in cell viability. The increase in cytosolic calcium induced by beta-amyloid is effectively blocked by cholesterol in a dose-dependent manner. Furthermore, pretreatment of PC12 cells with cholesterol also significantly attenuates the neurotoxicity induced by beta-amyloid. These findings suggest that extracellular free cholesterol can protect neurones from beta-amyloid neurotoxicity mediated by the disruption of calcium homeostasis.

Amyloid beta-Protein Precursor↗