Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.     

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.     

Molecular architecture of a multifunctional MCM complex

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.     

Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS

The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.     

Combining two teaching techniques for young children on Aedes aegypti control: effects on entomological indices in western Mexico

A study of the effect of educating four- to six-year-old children in mosquito control was recently conducted in a city in the state of Jalisco, western Mexico. Four neighborhood districts were selected. Children attending one kindergarten in each of two experimental districts were taught mosquito control with a video from the American Mosquito Control Association (AMCA), joined to the use of the AMCA Touch Table Technique. The entomological indices monitored in the study decreased significantly (P<0.05) in houses in the experimental districts, apparently because parents acted on the comments and suggestions of the children and eliminated or monitored containers used as oviposition sites by mosquitoes. Based on these results, combining both techniques for teaching children mosquito control is a potentially useful tool for control efforts in Mexico and other places in Latin America.     

Adaptation of microglomerular complexes in the honeybee mushroom body lip to manipulations of behavioral maturation and sensory experience

Worker honeybees proceed through a sequence of tasks, passing from hive and guard duties to foraging activities. The underlying neuronal changes accompanying and possibly mediating these behavioral transitions are not well understood. We studied changes in the microglomerular organization of the mushroom bodies, a brain region involved in sensory integration, learning, and memory, during adult maturation. We visualized the MB lips' microglomerular organization by applying double labeling of presynaptic projection neuron boutons and postsynaptic Kenyon cell spines, which form microglomerular complexes. Their number and density, as well as the bouton volume, were measured using 3D-based techniques. Our results show that the number of microglomerular complexes and the bouton volumes increased during maturation, independent of environmental conditions. In contrast, manipulations of behavior and sensory experience caused a decrease in the number of microglomerular complexes, but an increase in bouton volume. This may indicate an outgrowth of synaptic connections within the MB lips during honeybee maturation. Moreover, manipulations of behavioral and sensory experience led to adaptive changes, which indicate that the microglomerular organization of the MB lips is not static and determined by maturation, but rather that their organization is plastic, enabling the brain to retain its synaptic efficacy.     

In situ determination of a PKA phosphorylation site in the C-terminal region of filamin

A C-terminal region of human endothelial actin-binding protein-280 (ABP-280 or ABP, non-muscle filamin) was subcloned and efficiently expressed in a mammalian cells system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis. As predicted by the aminoacid sequence, the fragment, a 79 kD peptide (residues 1671–2361, plus 3.9 kD from an N-terminal fusion peptide included in the expression plasmid), contained the two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336) predicted to be present in this region of the molecule. Incubation of cells in the presence of cAMP-elevating agents enhanced ³²P uptake into the fragment. Site-directed mutagenesis analysis indicated that serine 2152 is the unique substrate in the C-terminal region of ABP for endogenously activated PKA. The functional implications of phosphorylation of this residue, which belongs to a serine-proline motif, are discussed in terms of the role of filamin in cytoskeleton reorganization.     

CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor

The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg ml^-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml^-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics of the mAb harvested from the bioreactor during the 43 days of cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.