Creation of salt-insensitive 3'(2'),5'-bisphosphate nucleotidase by modeling and mutagenesis approach

3′(2′),5′-Bisphosphate nucleotidase, (EC 3.1.3.7) (BPntase) is a ubiquitous enzyme. Recently, these enzymes have drawn considerable attention as in vivo targets of salt toxicity as well as therapeutic targets of lithium that is used for the treatment of manic-depressive disorders. They belong to the Mg²⁺-dependent Li⁺-sensitive phosphomonoesterase super-family and are highly sensitive to lithium and sodium ions. However, the molecular mechanism of inhibition of this group of enzymes by monovalent cations has not been completely understood. Previously we have identified a BPntase (Dhal2p) from a highly halotolerant yeast Debaryomyces hansenii. Molecular characterization revealed a number of unique features in Dhal2p, indicating this is an extraordinary member of the family. In this study, we have carried out the structure-function analysis of Dhal2p through the combination of molecular modeling and in vitro mutagenesis approach. We have not only provided the explanation for the role played by the functionally important elements that are conserved among the members of this family but also identified important, novel structural elements in this enzyme. Our study for the first time unraveled the role of a flap as well as a loop region in the functioning of this enzyme. Most importantly, mutations in the loop region resulted in the creation of a BPntase that was insensitive to salt.     

Photoperiodic effect on some enzymes and metabolites in diapausingAntheraea mylitta pupae andPhilosamia ricini larvae during development

The variation in acid phosphatase (EC 3.1.3.2) activity inAntheraea mylitta was similar in all light and dark groups exposed to different photophases (LD =0:24, 24:0, 18:6, 14:10, 10:14 and 12:12 h) maintaining all along a higher activity than its alkaline counterpart. The highest activity was recorded on day 82 in LD group 10:14 h. The non-diapausingPhilosamia ricini larvae registered highest activity in LD group 0:24 h on day 5. Alkaline phosphatase (EC 3.1.3.1) activity was low all through metamorphosis in both the lepidopterans, although significantly elevated activity was observed on day 5 in larvae of allPhilosamia ricini LD regimens and on day 82 inAntherae mylitta. Photoperiodic effect on Phosphorylase (EC 2.3.1.1) activity, glycogen and inorganic phosphates content have also been studied. Exposure to LD 10:14, 14:10 and 18:6 h provoked early diapause termination inAntheraea mylitta. The non-diapausingPhilosamia ricini was unaffected in moth emergence but the emerged adults of LD 24:0 and 0:24 h groups were unhealthy, small and did not mate or oviposit.     

Radiation-induced volatile hydrocarbon production in platelets

Generation of volatile hydrocarbons (ethane, pentane) as a measure of lipid peroxidation was followed in preparations from platelet-rich plasma irradiated in vitro. The hydrocarbons in the headspace of sealed vials containing irradiated and nonirradiated washed platelets, platelet-rich plasma, or platelet-poor plasma increased with time. The major hydrocarbon, pentane, increased linearly and significantly with increasing log radiation dose, suggesting that reactive oxygen species induced by ionizing radiation result in lipid peroxidation. Measurements of lipid peroxidation products may give an indication of suboptimal quality of stored and/or irradiated platelets.     

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD^−/− mice. LCAD^−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD^−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD^−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD^−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.     

Small molecule scaffolds that disrupt the Rev1-CT/RIR protein-protein interaction

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows replicative bypass of DNA lesions, including DNA adducts formed by cancer chemotherapeutics. Previous studies demonstrated that suppression of TLS can increase sensitivity of cancer cells to first-line chemotherapeutics and decrease mutagenesis linked to the onset of chemoresistance, marking the TLS pathway as an emerging therapeutic target. TLS is mediated by a heteroprotein complex consisting of specialized DNA polymerases, including the Y-family DNA polymerase Rev1. Previously, we developed a screening assay to identify the first small molecules that disrupt the protein–protein interaction between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) present in multiple DNA polymerases involved in TLS. Herein we report additional hit scaffolds that inhibit this key TLS PPI. In addition, through a series of biochemical, computational, and cellular studies we have identified preliminary structure–activity relationships and determined initial pharmacokinetic parameters for our original hits.     

Human mitochondrial function during cardiac growth and development

Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.     

Circadian rhythm in acetylcholinesterase activity during aging of the central nervous system

The circadian rhythm of acetylcholinesterase (AChE) activity was investigated in the cerebrum, cerebellum, medulla and optic lobes of rats aged 1 day and 3, 13, 44 and 87 weeks. The rhythm was found to be age-dependent. Animals aged 1 day exhibited a bimodal rhythm in all the four regions of the brain studied. At 3 and 13 weeks the activity was unimodal. The peak occurred during the light-on phase at 3 weeks and during the light-off phase at 13 weeks. At 87 weeks the rhythms in the medulla and cerebrum were similar to those of 44 week animals. By contrast the cerebellum had a bimodal rhythm with peaks at intervals of 12 h. In the optic lobes there was a shift from a bimodal pattern at 44 weeks to a unimodal one at 87 weeks. The times of onset of the light-on and light-off peaks in different regions of the brain differed with age.     

Study of the Interaction between Bacteriophage T4 asiA and Escherichia coli ς70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity to E. coli Cells by Coexpression of a Truncated ς70 Fragment

The interaction of T4 phage-encoded anti-sigma factor, asiA, and Escherichia coli sigma(70) was studied by using the yeast two-hybrid system. Truncation of sigma(70) to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased beta-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated sigma(70) fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated sigma(70) and asiA could be expressed in large quantities and purified.