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.

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.

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.

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.

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.

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.

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.

Effect of pectin, gum arabic and agar on cholesterol absorption, synthesis, and turnover in rats.

A series of five experiments was conducted to determine the effect of pectin, gum arabic and agar (5%) on cholesterol absorption, biosynthesis and turnover in rats. In the study of cholesterol absorption, a tracer dose of labeled cholesterol was included in the last meal. The rats were killed 12 hours later. The proportion of the labeled cholesterol recovered in the whole body was used as an estimation of the efficiency of absorption of dietary cholesterol. Cholesterol biosynthesis was estimated by determining the activity of labeled digitonin-precipitable sterols biosynthesized from labeled glucose which was included in a test meal. In turnover studies, rats were injected intravenously with labeled cholesterol using serum as a vehicle, and the activity of labeled cholesterol in tissues was determined after various time intervals. All three complex carbohydrates decreased cholesterol absorption and pectin had the greatest effect. Pectin and gum arabic increased cholesterol biosynthesis in rats fed a cholesterol-containing diet, but had no effect in a cholesterol-free diet. Pectin slightly increased the turnover of cholesterol, but gum arabic and agar had no effect. This work supports the hypothesis that pectin lowers cholesterol levels by interfering with cholesterol absorption and by increasing cholesterol turnover. The study also suggests that complex carbohydrates differ in their effects on cholesterol metabolism. The reason for these differences remains to be determined.