Calculations of intrinsic isotope effects and the detection of tunneling in enzyme-catalyzed reactions

The equation of Northrop [1975, Biochemistry, 14, 2644] for calculating intrinsic isotope effects from observed deuterium and tritium isotope effects of V/K, in which hydrogen is the reference isotope, has been extended to experimental designs using either deuterium or tritium as a reference. Partial derivatives of the intrinsic equations allow calculation of the relative precision of the three referenced isotope effects and these favor the order deuterium > tritium > hydrogen. In comparisons of observed and calculated isotope effects when hydrogen tunneling is present, both the precision and the magnitude of the difference was greater for intrinsic calculations than for exponentiations based upon a breakdown in the Swain-Schaad relationship.     

Effects of cloprostenol sodium and clenbuterol HCl on reproductive performance in postpartum anestrous cows

Two experiments were conducted to study effects of cloprostenol sodium (cloprostenol) and clenbuterol HCl (clenbuterol) during postpartum anestrus on subsequent reproductive performance in cows. In Experiment I, 96 cows received either 0.5 mg cloprostenol (PGF, n = 25), 364 mg clenbuterol (CLEN, n = 24), 0.5 mg cloprostenol and 364 mg clenbuterol (CLEN+PGF, n = 21) or no treatment (Control, n = 26) on Day 20 post partum. Treatments failed to influence postpartum interval, pregnancy rate or the incidence of short estrous cycles preceding the first normal estrous cycle. In Experiment II, anestrous cows were administered cloprostenol (0.5 mg) on either Day 20 (PGF20, n = 27) or Day 35 post partum (PGF35, n = 25), or served as untreated controls (Control, n = 26). Neither postpartum interval nor pregnancy rate were affected by cloprostenol treatment. In conclusion, treatment of postpartum cows with PGF did not alter the resumption of normal estrous cycles following parturition.     

Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.

We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs.     

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.     

Determinants of predation on phytophagous insects: the importance of diet breadth

To evaluate the role of predation in the evolution of diet specialization and to determine the effectiveness of various larval defenses, we offered lepidopteran larvae to colonies of the tropical ant Paraponera clavata. We recorded behavioral and physical characteristics of prey items and used log-linear models to analyze their importance as deterrents to predation by P. clavata. The most important determinant of probability of prey rejection by P. clavata was a prey's diet breadth; specialists were rejected by the ants significantly more than generalists. Other less important, but significant, predictors of prey rejection included ontogeny, morphology and chemistry. Late instar caterpillars were rejected more frequently than early instars, hairy caterpillars were rejected more frequently than caterpillars with other morphologies, and one caterpillar species with an unpalatable extract was rejected more frequently than two species with palatable extracts.     

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from -carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an -helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core -barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isolecuine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressedin vivo.