Progress toward chemical accuracy in the computer simulation of condensed phase reactions.

We describe a procedure for the generation of chemically accurate computer-simulation models to study chemical reactions in the condensed phase. The process involves (i) the use of a coupled semiempirical quantum and classical molecular mechanics method to represent solutes and solvent, respectively; (ii) the optimization of semiempirical quantum mechanics (QM) parameters to produce a computationally efficient and chemically accurate QM model; (iii) the calibration of a quantum/classical microsolvation model using ab initio quantum theory; and (iv) the use of statistical mechanical principles and methods to simulate, on massively parallel computers, the thermodynamic properties of chemical reactions in aqueous solution. The utility of this process is demonstrated by the calculation of the enthalpy of reaction in vacuum and free energy change in aqueous solution for a proton transfer involving methanol, methoxide, imidazole, and imidazolium, which are functional groups involved with proton transfers in many biochemical systems. An optimized semiempirical QM model is produced, which results in the calculation of heats of formation of the above chemical species to within 1.0 kcal/mol (1 kcal = 4.18 kJ) of experimental values. The use of the calibrated QM and microsolvation QM/MM (molecular mechanics) models for the simulation of a proton transfer in aqueous solution gives a calculated free energy that is within 1.0 kcal/mol (12.2 calculated vs. 12.8 experimental) of a value estimated from experimental pKa values of the reacting species.

Automated parallel DNA sequencing on multiple channel microchips

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Sensitivity to inhibition by β-chemokines correlates with biological phenotypes of primary HIV-1 isolates

Primary HIV-1 isolates were evaluated for their sensitivity to inhibition by β-chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. Virus isolates of both nonsyncytium-inducing (NSI) and syncytium-inducing (SI) biological phenotypes recovered from patients at various stages of HIV-1 infection were assessed, and the results indicated that only the isolates with the NSI phenotype were substantially inhibited by the β-chemokines. More important to note, these data demonstrate that resistance to inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β is not restricted to T cell line-adapted SI isolates but is also a consistent property among primary SI isolates. Analysis of isolates obtained sequentially from infected individuals in whom viruses shifted from NSI to SI phenotype during clinical progression exhibited a parallel loss of sensitivity to β-chemokines. Loss of virus sensitivity to inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β was furthermore associated with changes in the third variable (V3) region amino acid residues previously described to correlate with a shift of virus phenotype from NSI to SI. Of interest, an intermediate V3 genotype correlated with a partial inhibition by the β-chemokines. In addition, we also identified viruses sensitive to RANTES, MIP-1α, and MIP-1β of NSI phenotype that were isolated from individuals with AIDS manifestations, indicating that loss of sensitivity to β-chemokine inhibition and shift in viral phenotype are not necessarily prerequisites for the pathogenesis of HIV-1 infection.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

ParAlign: a parallel sequence alignment algorithm for rapid and sensitive database searches

There is a need for faster and more sensitive algorithms for sequence similarity searching in view of the rapidly increasing amounts of genomic sequence data available. Parallel processing capabilities in the form of the single instruction, multiple data (SIMD) technology are now available in common microprocessors and enable a single microprocessor to perform many operations in parallel. The ParAlign algorithm has been specifically designed to take advantage of this technology. The new algorithm initially exploits parallelism to perform a very rapid computation of the exact optimal ungapped alignment score for all diagonals in the alignment matrix. Then, a novel heuristic is employed to compute an approximate score of a gapped alignment by combining the scores of several diagonals. This approximate score is used to select the most interesting database sequences for a subsequent Smith–Waterman alignment, which is also parallelised. The resulting method represents a substantial improvement compared to existing heuristics. The sensitivity and specificity of ParAlign was found to be as good as Smith–Waterman implementations when the same method for computing the statistical significance of the matches was used. In terms of speed, only the significantly less sensitive NCBI BLAST 2 program was found to outperform the new approach. Online searches are available at http://dna.uio.no/search/

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution.

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.

Microevolutionary responses in experimental populations of plants to CO2-enriched environments: parallel results from two model systems.

Despite the critical role that terrestrial vegetation plays in the Earth's carbon cycle, very little is known about the potential evolutionary responses of plants to anthropogenically induced increases in concentrations of atmospheric CO2. We present experimental evidence that rising CO2 concentration may have a direct impact on the genetic composition and diversity of plant populations but is unlikely to result in selection favoring genotypes that exhibit increased productivity in a CO2-enriched atmosphere. Experimental populations of an annual plant (Abutilon theophrasti, velvetleaf) and a temperate forest tree (Betula alleghaniensis, yellow birch) displayed responses to increased CO2 that were both strongly density-dependent and genotype-specific. In competitive stands, a higher concentration of CO2 resulted in pronounced shifts in genetic composition, even though overall CO2-induced productivity enhancements were small. For the annual species, quantitative estimates of response to selection under competition were 3 times higher at the elevated CO2 level. However, genotypes that displayed the highest growth responses to CO2 when grown in the absence of competition did not have the highest fitness in competitive stands. We suggest that increased CO2 intensified interplant competition and that selection favored genotypes with a greater ability to compete for resources other than CO2. Thus, while increased CO2 may enhance rates of selection in populations of competing plants, it is unlikely to result in the evolution of increased CO2 responsiveness or to operate as an important feedback in the global carbon cycle. However, the increased intensity of selection and drift driven by rising CO2 levels may have an impact on the genetic diversity in plant populations.