Enhanced seasonal exchange of CO2 by northern ecosystems since 1960.

Seasonal variations of atmospheric carbon dioxide (CO2) in the Northern Hemisphere have increased since the 1950s, but sparse observations have prevented a clear assessment of the patterns of long-term change and the underlying mechanisms. We compare recent aircraft-based observations of CO2 above the North Pacific and Arctic Oceans to earlier data from 1958 to 1961 and find that the seasonal amplitude at altitudes of 3 to 6 km increased by 50% for 45° to 90°N but by less than 25% for 10° to 45°N. An increase of 30 to 60% in the seasonal exchange of CO2 by northern extratropical land ecosystems, focused on boreal forests, is implicated, substantially more than simulated by current land ecosystem models. The observations appear to signal large ecological changes in northern forests and a major shift in the global carbon cycle.

Effects of haloperidol on cholinergic striatal interneurons: relationship to oral dyskinesias.

In a subset of rats, typical antipsychotic drugs (tAPD) produce oral dyskinesias called vacuous chewing movements (VCMs) that resemble tardive dyskinesia (TD), a behavioral side effect seen in a subset of people following tAPD treatment. Morphological changes within the striatum following tAPD have been correlated to VCMs in animal models. The cholinergic system has been implicated in expression of TD. To test the hypothesis that the striatal cholinergic system is perturbed after haloperidol treatment, rats were administered haloperidol for three weeks and tested for VCMs; the striata were then processed for the immunocytochemical localization of choline-acetyltransferase (ChAT). Neuronal density measures of ChAT-labeled neurons showed a 22% decrease in haloperidol-treated versus controls rats and a 37% reduction in the lateral portion of the striatum only in rats with VCMs. These findings further support evidence of the possible involvement of the cholinergic system and the ventrolateral striatum in VCMs, and possibly TD.

Temporal variations of natural and anthropogenic radionuclides in sea otter skull tissue in the North Pacific Ocean.

Marine mammals being among the top predators in the food web tend to accumulate organic and inorganic contaminants from the environment. The body burden of contaminants in these species could reflect their foods and thus contaminant levels could serve as proxies on the changes of ecosystem. A pilot study was carried out to investigate the possibility of radionuclide leakage at Amchitka using a suite of sea otter (Enhydra lutris) skulls collected near Amchitka nuclear test-sites before (1950s) and after the testing (1990s), and at Adak, another Aleutian Island, about 300 km from Amchitka, where the potential impact of radionuclide leakage from Amchitka is expected to be negligible. In addition, the naturally occurring and anthropogenic radionuclide content on the sea otter skull was also utilized to investigate if there was any significant ecosystem changes in the environment. Concentration of 210Pb in sea otter bones collected during the 1950s was significantly higher than those collected in the 1990s. We propose that among the various factors that could cause this higher enrichment in 210Pb, changes in the sea otter prey is the most likely one. Comparison of the 137Cs, 90Sr, 239,240Pu concentrations appear not to be significantly higher in sea otter skulls collected in 1990s from Amchitka where the underground tests in 1965-71 than those from Adak, although significant differences were detected among different groups collected at various times.

Reducing mass degeneracy in SAR by MS by stable isotopic labeling.

Mass spectrometry (MS) promises to be an invaluable tool for functional genomics, by supporting low-cost, high-throughput experiments. However, large-scale MS faces the potential problem of mass degeneracy---indistinguishable masses for multiple biopolymer fragments (e.g., from a limited proteolytic digest). This paper studies the tasks of planning and interpreting MS experiments that use selective isotopic labeling, thereby substantially reducing potential mass degeneracy. Our algorithms support an experimental--computational protocol called structure-activity relation by mass spectrometry (SAR by MS) for elucidating the function of protein-DNA and protein-protein complexes. SAR by MS enzymatically cleaves a crosslinked complex and analyzes the resulting mass spectrum for mass peaks of hypothesized fragments. Depending on binding mode, some cleavage sites will be shielded; the absence of anticipated peaks implicates corresponding fragments as either part of the interaction region or inaccessible due to conformational change upon binding. Thus, different mass spectra provide evidence for different structure--activity relations. We address combinatorial and algorithmic questions in the areas of data analysis (constraining binding mode based on mass signature) and experiment planning (determining an isotopic labeling strategy to reduce mass degeneracy and aid data analysis). We explore the computational complexity of these problems, obtaining upper and lower bounds. We report experimental results from implementations of our algorithms.

The NOESY jigsaw: automated protein secondary structure and main-chain assignment from sparse, unassigned NMR data.

High-throughput, data-directed computational protocols for Structural Genomics (or Proteomics) are required in order to evaluate the protein products of genes for structure and function at rates comparable to current gene-sequencing technology. This paper presents the JIGSAW algorithm, a novel high-throughput, automated approach to protein structure characterization with nuclear magnetic resonance (NMR). JIGSAW applies graph algorithms and probabilistic reasoning techniques, enforcing first-principles consistency rules in order to overcome a 5-10% signal-to-noise ratio. It consists of two main components: (1) graph-based secondary structure pattern identification in unassigned heteronuclear NMR data, and (2) assignment of spectral peaks by probabilistic alignment of identified secondary structure elements against the primary sequence. Deferring assignment eliminates the bottleneck faced by traditional approaches, which begin by correlating peaks among dozens of experiments. JIGSAW utilizes only four experiments, none of which requires 13C-labeled protein, thus dramatically reducing both the amount and expense of wet lab molecular biology and the total spectrometer time. Results for three test proteins demonstrate that JIGSAW correctly identifies 79-100% of alpha-helical and 46-65% of beta-sheet NOE connectivities and correctly aligns 33-100% of secondary structure elements. JIGSAW is very fast, running in minutes on a Pentium-class Linux workstation. This approach yields quick and reasonably accurate (as opposed to the traditional slow and extremely accurate) structure calculations. It could be useful for quick structural assays to speed data to the biologist early in an investigation and could in principle be applied in an automation-like fashion to a large fraction of the proteome.

Reducing mass degeneracy in SAR by MS by stable isotopic labeling.

Mass spectrometry (MS) promises to be an invaluable tool for functional genomics, by supporting low-cost, high-throughput experiments. However, large-scale MS faces the potential problem of mass degeneracy--indistinguishable masses for multiple biopolymer fragments (e.g. from a limited proteolytic digest). This paper studies the tasks of planning and interpreting MS experiments that use selective isotopic labeling, thereby substantially reducing potential mass degeneracy. Our algorithms support an experimental-computational protocol called Structure-Activity Relation by Mass Spectrometry (SAR by MS), for elucidating the function of protein-DNA and protein-protein complexes. SAR by MS enzymatically cleaves a crosslinked complex and analyzes the resulting mass spectrum for mass peaks of hypothesized fragments. Depending on binding mode, some cleavage sites will be shielded; the absence of anticipated peaks implicates corresponding fragments as either part of the interaction region or inaccessible due to conformational change upon binding. Thus different mass spectra provide evidence for different structure-activity relations. We address combinatorial and algorithmic questions in the areas of data analysis (constraining binding mode based on mass signature) and experiment planning (determining an isotopic labeling strategy to reduce mass degeneracy and aid data analysis). We explore the computational complexity of these problems, obtaining upper and lower bounds. We report experimental results from implementations of our algorithms.

Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA.

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.

A PCR-based method for uniform 13C/15N labeling of long DNA oligomers.

A polymerase chain reaction (PCR)-based method is described for uniform 13C/15N labeling of DNA duplexes. In this method, multiple copies of a blunt-ended duplex are cloned into a plasmid with each copy containing the sequence of interest and the restriction HincII sequences at the 5' and 3' ends. PCR with uniformly 13C/15N-labeled dNTP precursors results in a labeled DNA duplex containing multiple copies of the sequence of interest. Use of bi-directional primers, instead of self-priming [Louis et al. (1998) J. Biol. Chem. 273, 2374-2378], produces a DNA fragment of unique length. Twenty-four cycles of PCR of this purified product followed by restriction and purification gives (with 30% yield) the uniformly 13C/15N-labeled duplex sequence for multi-nuclear magnetic resonance spectroscopy.

Overexpression, purification, and biophysical characterization of the heterodimerization domain of the core-binding factor beta subunit.

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.

The effect of chronic haloperidol treatment on dendritic spines in the rat striatum.

Previous studies have shown that schizophrenics, in comparison to controls, have reduced cortical spine density and smaller striatal spines. The current study in the rat was conducted to determine whether such differences could result from chronic neuroleptic treatment and whether they are correlated with neuroleptic-induced oral dyskinesias. Rats administered 1.5 mg/kg/day of haloperidol (HA) (n = 28) or water (n = 10) were tested for vacuous chewing movements (VCMs). After 6 months, rats were divided into low and high VCM groups; all but seven high VCM rats were sacrificed. These rats (withdrawn group) were withdrawn from HA for 4 weeks. Random electron micrographs of the striatum were analyzed for spine changes. Spine size was not significantly affected by HA (0.193 vs 0.174 microm2, HA and control, respectively) nor correlated with oral dyskinesias (0.191 vs 0.196 microm2, low and high VCM groups, respectively). These results suggest that smaller spines in schizophrenic striatum may be correlated with the disease rather than caused by neuroleptic treatment. Spine density decreased in the HA-treated group (32.7 +/- 9.5) in comparison to controls (53.7 +/- 7.3, P < 0.001) and remained low in the withdrawn group (35.0 +/- 4.2, P < 0.01). Spine density also decreased in both the low (37.3 +/- 9.9, P < 0.01) and the high (28.0 +/- 7.0, P < 0.000) VCM groups in comparison to controls. However, there was no significant difference between high and low VCM groups, suggesting that decreased spine density is independent of oral dyskinesias. These results suggest that the decreased spine density observed in schizophrenic cortex may be a result of neuroleptic treatment.

Comparison of backbone dynamics of reduced and oxidized Escherichia coli glutaredoxin-1 using 15N NMR relaxation measurements.

NMR-based structure determination of Escherichia coli glutaredoxin-1 in its reduced and oxidized forms revealed only subtle structural differences between the two forms. In an effort to characterize the role dynamics may play in the functioning of the protein, the backbone dynamics of both the reduced and oxidized forms of E. coli glutaredoxin-1 have been characterized using inverse-detection two-dimensional 15N-1H NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state [1H]-15N NOEs were measured for a majority of the protonated backbone nitrogen atoms. These data were analyzed by using a model-free formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau(e)), 15N exchange broadening contributions (R(ex)), and the overall molecular rotational correlation time (tau(m)). Sedimentation equilibrium measurements showed the reduced protein to be monomeric whereas the oxidized form could be fit to a monomer-dimer equilibrium. In order to try and assess the effect of dimerization on the dynamical parameters, the measurements on the oxidized protein have been carried out at two concentrations with very different monomer/dimer ratios. There is increased motion on both nano-picosecond and micro-millisecond time scales in the reduced form relative to the oxidized form, consistent with a more rigid oxidized protein. The increase in motion in the reduced protein correlates with its decreased thermodynamic stability. The role of the observed differences in the dynamic behavior in the two forms, particularly in the active site, in glutaredoxin-1's role as a protein disulfide reductant is discussed.

Identity of Escherichia coli D-1-amino-2-propanol:NAD+ oxidoreductase with E. coli glycerol dehydrogenase but not with Neisseria gonorrhoeae 1,2-propanediol:NAD+ oxidoreductase.

The properties of D-1-amino-2-propanol oxidoreductase from wild-type Escherichia coli have been compared with those of a glycerol dehydrogenase from mutant E. coli 424 and of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae. Several independent lines of evidence indicate that the former two enzymes are identical. (i) Both enzymatic activities purified to virtual homogeneity in an identical manner, and the ratio of specific activities (glycerol/aminopropanol) remained constant at all stages. (ii) When electrophoresed, both purified enzymes showed a major as well as a minor band of protein coincident with activity, and these two bands from each enzyme had the same mobility. (iii) The subunit molecular weights and isoelectric points were identical for each enzyme, and (iv) kinetic constants (Km and Vmax values) determined with three different substrates were the same. The somewhat greater stability of the glycerol dehydrogenase to controlled heat denaturation at 74 degrees C was the only difference observed between these two enzymes. In contrast, D-1-amino-2-propanol oxidoreductase was found to be immunochemically and kinetically distinct from the 1,2-propanediol oxidoreductase from N. gonorrhoeae.

D-1-amino-2-propanol:NAD+ oxidoreductase. Purification and general properties of the large molecular form of the enzyme from Escherichia coli K12.

Growth of Escherichia coli K12 under relatively anaerobic conditions in a medium containing casein hydrolysate, 0.8% glycerol, and 0.8% hydroxyacetone has been found to induce the level of D-1-amino-2-propanol oxidoreductase activity 50- to 100-fold over that in cells grown in casein hydrolysate alone or with 0.8% glycerol added. A large molecular weight form of this oxidoreductase (designated Form L) has been purified to apparent homogeneity in good yield by three simple steps designed to obviate its conversion to a smaller species. The molecular weight of native Form L and its basic subunit are 417,000 +/- 20,700 and 50,500 +/- 2,770, respectively; hence Form L would appear to consist of eight identical subunits. The pH activity profile for Form L shows one optimum in the range of 8.3 to 8.6 and another at pH 10.0 to 10.2. This form of the oxidoreductase has no apparent requirement for added metal ions (rather, numerous divalent transition metal ions are strongly inhibitory) or thiol compounds; it catalyzes the oxidation of several vic-glycols but is completely stereospecific for the D-isomer of 1-amino-2-propanol, utilizes only NAD+ as cosubstrate in the oxidation reaction (Km for NAD+ with DL-1-amino-2-propanol = 1.23 mM), but both NADH and NADPH serve as cosubstrate in the reduction of hydroxyacetone. Oxidoreductase activity of Form L is highly sensitive to inhibition by Hg2+, p-mercuribenzoate, or dithiodipyridine; inhibition by the latter two compounds is completely reversed by adding a thiol in excess.

Management of combined segment disease.

Eighty-five of 148 inflow procedures were performed for combined segment disease. Our study shows that aortofemoral bypass is clinically and functionally superior to axillofemoral bypass in limbs with combined segment disease and hemodynamic criteria for limb salvage. The results of these two procedures are comparable for claudicant limbs. A derivative of segmental plethysmography, the predictive index, can select preoperatively those limbs that will fail to respond to aortofemoral bypass alone. Finally, either in limbs selected for aortofemoral bypass with both ischemic tissue lesions and a predictive index greater than 0.2 or in limbs selected for axillofemoral bypass with ischemic tissue lesions alone, a synchronous procedure can be performed with relatively low morbidity and excellent early functional results.