Influence of alpha-CH-->NH substitution in C8-CoA on the kinetics of association and dissociation of ligands with medium chain acyl-CoA dehydrogenase

We previously reported that the kinetic profiles for the association and dissociation of functionally diverse C(8)-CoA-ligands, viz., octanoyl-CoA (substrate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical, suggesting that the protein conformational changes played an essential role during ligand binding and/or catalysis [Peterson, K. L., Sergienko, E. E., Wu, Y., Kumar, N. R., Strauss, A. W., Oleson, A. E., Muhonen, W. W., Shabb, J. B., and Srivastava, D. K. (1995) Biochemisry 34, 14942-14953]. To ascertain the structural basis of the above similarity, we investigated the kinetics of association and dissociation of alpha-CH-->NH-substituted C(8)-CoA, namely, 2-azaoctanoyl-CoA, with the recombinant form of human liver MCAD. The rapid-scanning and single wavelength stopped-flow data for the binding of 2-azaoctanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involves the formation of an enzyme-ligand collision complex (with a dissociation constant of K(c)), followed by a slow isomerization step (with forward and reverse rate constants of k(f) and k(r), respectively) with concomitant changes in the electronic structure of the enzyme-bound FAD. Since the latter step involves a concurrent change in the enzyme's tryptophan fluorescence, it is suggested that the isomerization step is coupled to the changes in the protein conformation. Although the overall binding affinity (K(d)) of the enzyme-2-azaoctanoyl-CoA complex is similar to that of the enzyme-octenoyl-CoA complex, their microscopic equilibria within the collision and isomerized complexes show an opposite relationship. These results coupled with the isothermal titration microcalorimetric studies lead to the suggestion that the electrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as their corresponding ground and transition states, during the course of the enzyme-ligand interaction.     

Stereochemical outcome and kinetic effects of Rp- and Sp-phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase

To probe the mechanism of the reversible DNA phosphodiester bond cleavage and religation mechanism of the type I topoisomerase from vaccinia virus, we have synthesized DNA substrates carrying a single nonbridging Rp- or Sp-phosphorothioate (Ps) modification at the scissile phosphodiester (Pd) bond. Analysis of the stereochemical outcome of the net cleavage and rejoining reaction established that the reaction proceeds with retention of configuration, as expected for a double-displacement mechanism. Single-turnover kinetic studies on irreversible strand cleavage using 18/24 mer suicide substrates showed thio effects (k(Pd)/k(Ps)) of 340- and 30-fold for the Rp-Ps and Sp-Ps stereoisomers, respectively, but approximately 10-fold smaller thio effects for the reverse single-turnover religation reaction (Rp-Ps = 30 and Sp-Ps = 3). As compared to the smaller suicide cleavage substrates, approach-to-equilibrium cleavage studies using 32/32 mer substrates showed 7-9-fold smaller thio effects on cleavage, similar effects on religation, and the same ratio of the Rp to Sp thio effect as the suicide cleavage reaction ( approximately 10). In general, thio effects of 2.4-7.2-fold on the cleavage equilibrium are observed for the wild-type and H265A enzymes, suggesting differences in the interactions of the enzyme with the nonbridging sulfur in the noncovalent and covalent complexes. Studies of the cleavage, religation, and approach-to-equilibrium reactions catalyzed by the H265A active site mutant revealed a stereoselective, 11-fold decrease in the Rp-thio effect on cleavage and religation as compared to the wild-type enzyme. This result suggests that His-265 interacts with the nonbridging pro-Rp oxygen in the transition state for cleavage and religation, consistent with the arrangement of this conserved residue in the crystal structure of the human topoisomerase-DNA complex. In general, the greatest effect of thio substitution and the H265A mutation is to destabilize the transition state, with smaller effects on substrate binding. The interaction of His-265 with the pro-Rp nonbridging oxygen is inconsistent with the proposal that this conserved residue acts as a general acid in the strand cleavage reaction.     

B cell epitopes in the intrinsically disordered regions of neuraminidase and hemagglutinin proteins of H5N1 and H9N2 avian influenza viruses for peptide-based vaccine development

Avian influenza viruses (AIV) are very active in several parts of the globe and are the cause of huge economic loss for the poultry industry and also human fatalities. Three dimensional modeling was carried out for neuraminidase (NA) and hemagglutinin (HA) proteins of AIV. The C-score, estimated TM-Score, and estimated root-mean-square deviation (RMSD) score for NA of H5N1 were −1.18, 0.57 ± 0.15, and 9.8 ± 7.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for NA of H9N2 were −1.43, 0.54 ± 0.15, and 10.5 ± 4.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H5N1 were −0.03, 0.71 ± 0.12, and 7.7 ± 4.3, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H9N2 were −0.57, 0.64 ± 0.13, and 8.9 ± 4.6, respectively. Intrinsically disordered regions were identified for the NA and HA proteins of H5N1 and H9N2 with the use of PONDR program. Linear B cell epitope was predicted using BepiPred 2 program for NA and HA of H5N1 and H9N2 avian influenza strains. Discontinuous epitopes were predicted by Discotope 2 program. The linear epitopes that were considered likely to be immunogenic and within the intrinsically disordered region for the NA of H5N1 was TKSTNSRSGFEMIWDPNGWTGTDSSFSVK, and for H9N2 it was VGDTPRNDDSSSSSNCRDPNNERGAP. In the case of HA of H5N1, it was QRLVPKIATRSKVNGQSG and ATGLRNSPQRERRRKK; for H9N2 it was INRTFKPLIGPRPLVNGLQG and SLKLAVGLRNVPARSSR. The discontinuous epitopes of NA of H5N1 and H9N2 were identified at various regions of the protein structure spanning from amino acid residue positions 90 to 449 and 107 to 469, respectively. Similarly, the discontinuous epitopes of HA of H5N1 and H9N2 were identified in the amino acid residue positions 27 to 517 and 136 to 521, respectively. This study has identified potential and highly immunogenic linear and conformational B-cell epitopes towards developing a vaccine against AIV both for human and poultry use.     

Effects of tribromoethanol-medetomidine combination on cycling and ovariectomized Sprague-Dawley rats

Although commonly used to induce anesthesia in rodents, the effective dose of tribromoethanol is associated with various side effects. The authors previously found that a tribromoethanol-medetomidine combination reduced the dose of tribromoethanol necessary for effective anesthesia in male Sprague-Dawley rats, an effect reversible by atipamezole. Here, the authors focus on the effect of this anesthetic combination in female Sprague-Dawley rats, its effects on their estrous cycles, and its efficacy at low sex hormone levels. Their results suggest that the anesthetic combination is effective in female rats, does not affect their estrous cycles, and works even when hormone levels are low, such as after ovariectomy.     

A New Species of Microhyla (Anura: Microhylidae) from Nilphamari,Bangladesh

A new species of Microhyla frog from the Nilphamari district of Bangladesh is described and compared with its morphologically similar and geographically proximate congeners. Molecular phylogeny derived from mitochondrial DNA sequences revealed that although the new species – designated here as Microhyla nilphamariensis sp. nov. – forms a clade with M. ornate, it is highly divergent from M. ornata and all of its congeners, with 5.7 – 13.2% sequence divergence at the 16S rRNA gene. The new species can be identified phenotypically on the basis of a set of diagnostic (both qualitative and quantitative) characters as follows: head length is 77% of head width, distance from front of eyes to the nostril is roughly six times greater than nostril–snout length, internarial distance is roughly five times greater than nostril–snout length, interorbital distance is two times greater than internarial distance, and distance from back of mandible to back of the eye is 15% of head length. Furthermore, inner metacarpal tubercle is small and ovoid-shaped, whereas outer metacarpal tubercle is very small and rounded. Toes have rudimentary webbing, digital discs are absent, inner metatarsal tubercle is small and round, outer metatarsal tubercle is ovoid-shaped, minute, and indistinct.     

Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver

Plasma alpha-linolenic acid (alpha-LNA, 18:3n-3) and linoleic acid (LA, 18:2n-6) do not contribute significantly to the brain content of docosahexaenoic acid (DHA, 22:6n-3) or arachidonic acid (AA, 20:4n-6), respectively, and neither DHA nor AA can be synthesized de novo in vertebrate tissue. Therefore, measured rates of incorporation of circulating DHA and AA into brain exactly represent their rates of consumption by brain. Positron emission tomography (PET) has been used to show, based on this information, that the adult human brain consumes AA and DHA at rates of 17.8 and 4.6 mg/day, respectively, and that AA consumption does not change significantly with age. In unanesthetized adult rats fed an n-3 PUFA "adequate" diet containing 4.6% alpha-LNA (of total fatty acids) as its only n-3 PUFA, the rate of liver synthesis of DHA was more than sufficient to maintain brain DHA, whereas the brain's rate of DHA synthesis is very low and unable to do so. Reducing dietary alpha-LNA in the DHA-free diet led to upregulation of liver but not brain coefficients of alpha-LNA conversion to DHA and of liver expression of elongases and desaturases that catalyze this conversion. Concurrently, brain DHA loss slowed due to downregulation of several of its DHA-metabolizing enzymes. Dietary alpha-LNA deficiency also promoted accumulation of brain docosapentaenoic acid (22:5n-6), and upregulated expression of AA-metabolizing enzymes, including cytosolic and secretory phospholipases A(2) and cyclooxygenase-2. These changes, plus reduced levels of brain derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) in n-3 PUFA diet deficient rats, likely render their brain more vulnerable to neuropathological insults.