Moderate severity heart failure does not involve a downregulation of myocardial fatty acid oxidation

Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-(3)H]oleate, [U-(14)C]glucose, and [1-(13)C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomenon.     

Automated purification and suspension array detection of 16S rRNA from soil and sediment extracts by using tunable surface microparticles

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA ( approximately 10(4) cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA ( approximately 10(5) cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.     

The conformational states of Mg·ATP in water

The conformational equilibria of Mg·ATP in solution is studied using molecular dynamics (MD) augmented with umbrella sampling methods. Free energy comparisons show that the Mg²⁺ ion is equally likely to coordinate the oxygens of the two end phosphates, or of all three phosphates. The MD trajectories reveal two major degrees of freedom of the Mg·ATP molecule in solution, and we compute the free energy as a function of these variables, and determine its elastic properties. Comparing the free energy function with several crystallographic structures of ATP analogs, we find that the crystal structures correspond to states where ATP would be elastically strained. The average water density around Mg·ATP is investigated to show the average number of hydrogen bonds and the hydrophobicity.     

Enterococcal peptide sex pheromones: synthesis and control of biological activity

The enterococcal pheromone-inducible plasmids such as pCF10 represent a unique class of mobile genetic elements whose transfer functions are induced by peptide sex pheromones. These pheromones are excreted by potential recipient cells and detected by plasmid-containing donor cells at the cell surface, where the pheromone is imported and signals induction of the plasmid transfer system. Pheromone is processed from a chromosomally encoded lipoprotein and excreted by both the donor and recipient cells, but a carefully controlled detection system prevents a response to self-pheromone while still allowing an extremely sensitive response to exogenous pheromone.     

Production of huitlacoche, Ustilago maydis: timing inoculation and controlling pollination

Huitlacoche is the name given to young, fleshy, edible galls that form when ears of Zea mays are infected by Ustilago maydis. Huitlacoche is processed and sold fresh at markets in Mexico. Interest has increased recently in producing U. maydis as a specialty mushroom in the United States. Silk-channel inoculation methods developed to evaluate common smut resistance in maize can be used to produce huitlacoche commercially. This research assessed the effects of time of inoculation and preventing pollination on the severity of ear galls and yield of huitlacoche produced by inoculating silks with U. maydis. Yield of huitlacoche and severity of ear galls were closely related, as was evident from highly significant linear or curvilinear regressions. Severity and yield were greatest when ears were inoculated 4-8 d after the mid-silk growth stage. Ear galls were 5-15% more severe and yield of huitlacoche was 18-150% greater on ears that were not pollinated, compared to those that were pollinated. Maximum yield of huitlacoche was 131 g ear(-1) from unpollinated male-sterile field corn inoculated 6 d after the mid-silk growth stage and 92 g ear(-1) from detasseled sweet corn inoculated 6 d after mid-silk. About 25% of the total weight of ears consisted of marketable huitlacoche when yields were highest. Quality of huitlacoche was not affected by time of inoculation or pollination treatments, but quality of huitlacoche harvested 12-14 d after inoculation was unacceptable primarily due to lack of teliospores, which affected color and flavor.     

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.     

Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8(+) dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.