The Response to Exercise with Constant Energy Intake in Identical Twins

Seven pairs of young adult male identical twins completed a negative energy balance protocol during which they exercised on cycle ergometers twice a day, 9 out of 10 days, over a period of 93 days while being kept on a constant daily energy and nutrient intake. The total energy deficit caused by exercise above the estimated energy cost of body weight maintenance reached 244 ± 9.8 MJ (Mean ± SEM). Baseline energy intake was estimated over a period of 17 days preceding the negative energy balance protocol. Mean body weight loss was 5.0 kg (SEM = 0.6) (p <0.001) and it was entirely accounted for by the loss of fat mass (p <0.001). Fat-free mass was unchanged. Body energy losses reached 191 MJ (SEM = 24) (p <0.001) which represented about 78% of the estimated energy deficit. Subcutaneous fat loss was slightly more pronounced on the trunk than on the limbs as estimated from skinfolds, circumferences, and computed tomography (CT). The reduction in CT-assessed abdominal visceral fat was quite striking, from 81 cm² (SEM = 5) to 52 cm² (SEM = 6) (p <0.001). At the same submaximal power output level, subjects oxidized more lipids than carbohydrates after the program as indicated by the changes in the respiratory exchange ratio (p <0.05). Intrapair resemblance was observed for the changes in body weight (p <0.05), fat mass (P <0.01), percent fat (p <0.01), body energy content (p <0.01), sum of 10 skinfolds (p <0.01), abdominal visceral fat (p <0.01), fasting plasma triglycerides (p <0.05) and cholesterol (p <0.05), maximal oxygen uptake (p <0.05), and respiratory exchange ratio during submaximal work (p <0.01). We conclude that even though there were large individual differences in response to the negative energy balance and exercise protocol, subjects with the same genotype were more alike in responses than subjects with different genotypes particularly for body fat, body energy, and abdominal visceral fat changes. High lipid oxidizers and low lipid oxidizers during sub-maximal exercise were also seen despite the fact that all subjects had experienced the same exercise and nutritional conditions for about three months.     

Assessment of microinjection for introducing DNA into uninuclear microspores of rapeseed

Approximately 2,000 embryogenic uninuclear microspores of rapeseed (Brassica napus) cv. Topas were intranuclearly injected with a chimaeric -glucuronidase (Escherichia coli Uid A) gene. Stable integration had not occurred among 55 plants that were regenerated. Coinjection of the dye Lucifer Yellow and detection of injected DNA by the polymerase chain reaction revealed high frequencies of transfer. However, the amount of DNA injected was less than 20 copies, which may have been insufficient for stable transformation of microspores.Abbreviations PCR polymerase chain reaction - GUS -glucuronidase     

Actin microfilaments and fibroin secretion in the silkgland cells of Bombyx mori : Effects of cytochalasin B

Bombyx mori posterior silkgland cells exhibit an impressive microfilament apparatus located at the cellular apex. It consists of bundles of packed, long microfilaments of 50–70 Å diameter running along circumferences delimiting the lumen of the gland, perpendicularly to the flow of luminal silk. Microfilaments are closely associated with microtubules of the cytoplasmic ‘radial microtubule system’. Immunolabelling with purified antihuman actin antibodies was used to demonstrate their actin-like nature. Apical microfilaments are sensitive to cytochalasin B (CB) which selectively inhibits the secretion of fibroin. Following the removal of the drug, microfilaments recover their normal morphology and secretion resumes. The possible implication of contraction of microfilaments in the process of secretion is discussed.     

Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.     

Indolyl esters and amides related to indomethacin are selective COX-2 inhibitors

Previous studies from our laboratory have revealed that esterification/amidation of the carboxylic acid moiety in the nonsteroidal anti-inflammatory drug, indomethacin, generates potent and selective COX-2 inhibitors. In the present study, a series of reverse ester/amide derivatives were synthesized and evaluated as selective COX-2 inhibitors. Most of the reverse esters/amides displayed time-dependent COX-2 inhibition with IC₅₀ values in the low nanomolar range. Replacement of the 4-chlorobenzoyl group on the indole nitrogen with a 4-bromobenzyl moiety resulted in compounds that retained selective COX-2 inhibitory potency. In addition to inhibiting COX-2 activity in vitro, the reverse esters/amides also inhibited COX-2 activity in the mouse macrophage-like cell line, RAW264.7. Overall, this strategy broadens the scope of our previous methodology of neutralizing the carboxylic acid group in NSAIDs as a means of generating COX-2-selective inhibitors and is potentially applicable to other NSAIDs.