Comparative diffusion tractography of corticostriatal motor pathways reveals differences between humans and macaques.

The primate corticobasal ganglia circuits are understood to be segregated into parallel anatomically and functionally distinct loops. Anatomical and physiological studies in macaque monkeys are summarized as showing that an oculomotor loop begins with projections from the frontal eye fields (FEF) to the caudate nucleus, and a motor loop begins with projections from the primary motor cortex (M1) to the putamen. However, recent functional and structural neuroimaging studies of the human corticostriatal system report evidence inconsistent with this organization. To obtain conclusive evidence, we directly compared the pattern of connectivity between cortical motor areas and the striatum in humans and macaques in vivo using probabilistic diffusion tractography. In macaques we found that FEF is connected with the head of the caudate and anterior putamen, and M1 is connected with more posterior sections of the caudate and putamen, corroborating neuroanatomical tract tracing findings. However, in humans FEF and M1 are connected to largely overlapping portions of posterior putamen and only a small portion of the caudate. These results demonstrate that the corticobasal connectivity for the oculomotor and primary motor loop is not entirely segregated for primates at a macroscopic level and that the description of the anatomical connectivity of corticostriatal motor systems in humans does not parallel that of macaques, perhaps because of an expansion of prefrontal projections to striatum in humans.

Identification of butyrylcholinesterase adducts after inhibition with isomalathion using mass spectrometry: difference in mechanism between (1R)- and (1S)-stereoisomers.

Previous kinetic studies found that butyrylcholinesterase (BChE) inhibited by (1R)-isomalathions readily reactivated, while enzyme inactivated by (1S)-isomers did not. This study tested the hypothesis that (1R)- and (1S)-isomers inhibit BChE by different mechanisms, yielding distinct adducts identifiable by peptide mass mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Equine BChE (EBChE) was inhibited to <10% of control activity with each isomer of isomalathion and the reference compound isoparathion methyl. Control and treated enzyme was digested with trypsin, and peptides were fractionated with HPLC. Separated and unseparated peptides were analyzed with MALDI-TOF-MS. Identity of an organophosphorus peptide adduct was confirmed by fragmentation using postsource decay analysis. EBChE inhibited by (1R)-isomalathions or (S)-isoparathion methyl readily reactivated after oxime treatment with 30-40% activity recovered. Enzyme inactivated by (1S)-isomalathions or (R)-isoparathion methyl recovered <2% and <5% activity, respectively, after oxime treatment. MALDI-TOF-MS analysis revealed that inhibition of EBChE by (1R)-isomalathions and (R)- or (S)-isoparathion methyl yielded O,S-dimethyl phosphate adducts. Enzyme inactivated by (1S)-isomalathions produced only O-methyl phosphate adduct. EBChE modified by (1R)-isomalathions or either enantiomer of isoparathion methyl yielded an O-methyl phosphate adduct as well. The results indicate that EBChE inhibition by (1R)-isomalathions proceeds with loss of diethyl thiosuccinate, but inactivation by (1S)-isomers occurs with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate to yield an aged enzyme. Furthermore, the data suggest that aging of the O,S-dimethyl phosphate adduct occurs via an S(N)2 process with loss of thiomethyl.

C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping.

To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

Regional dendritic and spine variation in human cerebral cortex: a quantitative golgi study.

The present study explored differences in dendritic/spine extent across several human cortical regions. Specifically, the basilar dendrites/spines of supragranular pyramidal cells were examined in eight Brodmann's areas (BA) arranged according to Benson's (1993, Behav Neurol 6:75-81) functional hierarchy: primary cortex (somatosensory, BA3-1-2; motor, BA4), unimodal cortex (Wernicke's area, BA22; Broca's area, BA44), heteromodal cortex (supple- mentary motor area, BA6beta; angular gyrus, BA39) and supramodal cortex (superior frontopolar zone, BA10; inferior frontopolar zone, BA11). To capture more general aspects of regional variability, primary and unimodal areas were designated as low integrative regions; heteromodal and supramodal areas were designated as high integrative regions. Tissue was obtained from the left hemisphere of 10 neurologically normal individuals (M(age) = 30 +/- 17 years; five males, five females) and stained with a modified rapid Golgi technique. Ten neurons were sampled from each cortical region (n = 800) and evaluated according to total dendritic length, mean segment length, dendritic segment count, dendritic spine number and dendritic spine density. Despite considerable inter-individual variation, there were significant differences across the eight Brodmann's areas and between the high and low integrative regions for all dendritic and spine measures. Dendritic systems in primary and unimodal regions were consistently less complex than in heteromodal and supramodal areas. The range within these rankings was substantial, with total dendritic length in BA10 being 31% greater than that in BA3-1-2, and dendritic spine number being 69% greater. These findings demonstrate that cortical regions involved in the early stages of processing (e.g. primary sensory areas) generally exhibit less complex dendritic/spine systems than those regions involved in the later stages of information processing (e.g. prefrontal cortex). This dendritic progression appears to reflect significant differences in the nature of cortical processing, with spine-dense neurons at hierarchically higher association levels integrating a broader range of synaptic input than those at lower cortical levels.

Inhibition of acetylcholinesterase by (1S,3S)-isomalathion proceeds with loss of thiomethyl: kinetic and mass spectral evidence for an unexpected primary leaving group.

Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.

Continuous quality improvement for patients with back pain.

Recent evidence has changed traditional approaches to low back pain, suggesting minimal bed rest, highly selective imaging, and early return to normal activities. However, there are wide geographical variations in care, and substantial gaps between practice and evidence. This project sought to merge scientific evidence about back pain and knowledge about behavior change to help organizations improve care for back pain. Participating insurance plans, HMOs, and group practices focused on problems they themselves identified. The year-long program included quarterly meetings, coaching for rapid cycles of change, a menu of potential interventions, and recommendations for monitoring outcomes. Participants interacted through meetings, e-mail, and conference calls. Of the 22 participating organizations, 6 (27%) made major progress. Typical changes were reduced imaging, bed rest, and work loss, and increased patient education and satisfaction. Specific examples were a 30% decrease in plain x-rays, a 100% increase in use of patient education materials, and an 81% drop in prescribed bed rest. Despite the complexity of care for back pain, rapid improvements appear feasible. Several organizations had major improvements, and most experienced at least modest improvements. Key elements of successful programs included focus on a small number of clinical goals, frequent measurement of outcomes among small samples of patients, vigilance in maintaining gains; involvement of office staffs as well as physicians, and changes in standard protocols for imaging, physical therapy, and referral.

Photoexcited GaAs surfaces studied by transient terahertz time-domain spectroscopy.

The transmission characteristics of an air-GaAs interface and the transient absorption and index spectra of the thin, photoexcited surface layer are investigated subsequent to excitation by a femtosecond laser pulse. We find that the total phase change and transmission of a terahertz (THz) probe pulse are dominated by interface effects. This observation has important implications in the interpretation of THz time-domain spectroscopy data of absorbing media. We also observe that the THz pulse apparently arrives at the detector as much as 60 fs earlier when it is transmitted through an optically excited GaAs wafer. This effect is fully explained in terms of a frequency-dependent transmission and phase shift at the air-GaAs interface and is not associated with superluminal propagation.

Quality improvements in end of life care: insights from two collaboratives.

BACKGROUND: Just a few generations ago, most people died suddenly, at any age. Now, most die of serious chronic disease, after a substantial period of disability. The care system does not serve this burgeoning population well. However, two quality improvement (QI) collaboratives sponsored by the Institute for Healthcare Improvement and the Center to Improve Care of the Dying set about making substantial improvements. INSIGHTS GAINED: The participating organization teams in two Breakthrough Series collaboratives found it best to identify patients by asking "Would it be surprising for this patient to die in the next year? (or the next few months?)" All the teams used standard QI approaches, with an aim, measures, and changes to try in Plan-Do-Study-Act cycles. In the first collaborative, 42 (89%) of the 47 teams made important improvements in their care systems. Because of the strength of their changes, the high performance of their team, the administrative support they received, and their ability to partner with other agencies, 13 (27%) of the teams made substantial, measurable improvement during the collaborative. In the second collaborative, 29 (85%) of the 34 teams made key changes to their care system, and 16 (47%) of the teams made substantial, measurable improvement. Coordination across programs such as between a hospital and a long term care facility or hospice remained an elusive goal, and good care cannot become routine without financing and coverage reform. CONCLUSION: Clinical providers can reliably make substantial improvements in end of life care, within a few months, and within current financing and regulation. Coordinated efforts in two Breakthrough Series produced generalizable insights.

Evaluating predictors and concomitants of patient health visit satisfaction: an empirical study focusing on methodological aspects of satisfaction research.

We propose that the weak and conflicting results from various studies of satisfaction concomitants are due to the use of scales which sum scores from items that measure different types of satisfaction. The hypothesis is clearly supported, leading to the strong recommendation that different types of patient satisfaction be studied separately. Implications for studying and potentially increasing patient satisfaction are discussed.

High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts.

(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals. Limitations in enzyme supply from natural resources were overcome by production of the enzyme in the microbial host systems Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. Expression of Hnl in the prokaryotic system led to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein were obtained. Highest yields were obtained in a high cell density batch fermentation of a P. pastoris transformant that expressed heterologous Hnl to about 50% of the soluble cytosolic protein. At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product was obtained. Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies were not successful. Immunofluorescence microscopy studies indicated that the secretion-directed heterologous Hnl protein accumulated in the plasma membrane forming aggregated clusters of inactive protein.

Body temperature and wheel running predict survival times in rats exposed to activity-stress.

The relationship between restricted feeding, core body temperature (Tb), wheel running, survival, and gastric erosion formation was examined in female rats exposed to activity-stress. Core body temperature and gross motor activity were telemetrically monitored in four groups of rats that had free access to running wheels and in one group that was not allowed to run on the wheels. Twenty-four hours prior to the onset of hypothermia and predicted mortality, different groups were left undisturbed, warmed with a heat lamp, denied access to running wheels, or euthanized. Length of survival in wheel-running rats varied from 2 to 12 days. During the first day of food deprivation, premorbid changes in the variability of Tb during the diurnal period and the mean number of wheel revolutions during the nocturnal period were strongly predictive of length of survival. Warming rats with a heat lamp or preventing rats from ever running on the wheel increased the length of survival and attenuated gastric erosion formation. Only rats that were warmed had a greater likelihood of survival. Gastric pathology was also reduced in rats that were euthanized prior to becoming moribund. Rats that were left undisturbed or locked from the running wheel over the last 24 h of testing became moribund and had extensive gastric mucosal damage. These results indicate that thermoregulatory disturbances induced by restricted feeding and not wheel running alone are critical in determining survival and the degree of gastric mucosal injury in rats exposed to activity-stress. Results further suggest that predisposing factors may put some rats at risk for the development of activity-stress-induced mortality.

Life-span dendritic and spine changes in areas 10 and 18 of human cortex: a quantitative Golgi study.

Dendritic neuropil is a sensitive indicator of the aging process and may exhibit regional cortical variations. The present study examined regional differences and age-related changes in the basilar dendrites/spines of supragranular pyramidal cells in human prefrontal (area 10) and secondary occipital (area 18) cortices. Tissue was obtained from the left hemisphere of 26 neurologically normal individuals ranging in age from 14 to 106 years (M(age) = 57 +/- 22 years; 13 males, 13 females). In tissue prepared by a modified rapid Golgi technique, ten neurons were sampled from each cortical region (N = 520) and were evaluated according to the following parameters: total dendritic length, mean segment length, dendritic segment count, dendritic spine number, and dendritic spine density. The effects of age and Brodmann areas were analyzed with a nested multiple analysis of variance design. Despite considerable interindividual variation, several clear findings emerged: 1) Dendritic systems were significantly larger in area 10 than in area 18 across the sampled life span, presumably because of the more integrative function of area 10 neurons. 2) There was a significant age effect, with a substantial decline in dendritic neuropil from the younger (< or =50 years) group to the older (>50 years) group, especially in spine measures, which decreased almost 50%. 3) Dendritic values were relatively stable after 40 years of age, suggesting that dendritic/spine degeneration in older, relatively healthy individuals may not be an inevitable consequence of the aging process. These findings underscore the importance of life-long commitment to a cognitively invigorating environment.

Direct and indirect effects of demographic, medical, and psychological variables on neuropsychological performance in normal geriatric persons: a structural equation model.

The direct and indirect effects of demographic, medical, and psychological variables on neuropsychological performance in elderly individuals were examined using a LISREL structural equation model. One-hundred fifty-six geriatric subjects were individually administered a comprehensive neuropsychological battery, an extensive medical history and demographics questionnaire, and the Neuropsychology Behavior and Affect Profile (a psychological assessment instrument). The model assessed the effects of five independent latent variables (medical history, psychological functioning, global mental status, education, and gender-related functioning) on two dependent latent variables (nonverbal and verbal neuropsychological functioning). The best fitting model revealed that three latent variables (medical history, global mental status, and gender-related functioning) had direct effects on neuropsychological functioning and that all five independent variables exhibited indirect effects. These findings suggest that the influence of demographic variables on neuropsychological functioning for geriatric persons is complex and that certain variables should not be interpreted independently of each other due to their significant moderating influences.

Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis.

Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.

Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue.

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.