Simultaneous Retention of Thermostability and Specific Activity in Chimeric Human Alkaline Phosphatases

Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75 °C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization.     

Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA

HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA.     

Intrinsic prey suitability in specialist and generalist Harmonia ladybirds: a test of the trade‐off hypothesis for food specialization

The trade‐off hypothesis posits that increased performance on a given resource comes at the cost of decreased performance on other resources, and that this trade‐off is a driving force of food specialization in both predators and herbivores. In this study, we examined larval survival and performance in two sibling ladybird species, Harmonia yedoensis Takizawa and Harmonia axyridis Pallas (Coleoptera: Coccinellidae), fed on one of four prey species. Harmonia yedoensis is a specialist predator that preys mostly on pine aphids in the field, whereas H. axyridis is a generalist predator with a broad prey range. We experimentally showed in the laboratory that larval survival and performance were not higher when H. yedoensis was fed on pine aphids, compared with the other prey species. Rather, prey suitability was similar in both ladybird species, and H. yedoensis larvae developed as well or even better on prey species that they never utilize in nature. These results suggest that the host range in H. yedoensis may not be limited by the intrinsic suitability of the aphid species per se. Moreover, as shown by our previous study, the pine aphid is a highly elusive prey that is difficult for small ladybird hatchlings to capture, which means that the cost of utilizing this prey is high. Therefore, we conclude that some factor other than prey suitability is responsible for the observed food specialization in H. yedoensis.     

A Supply of Nitrogen Causes Increase in the Level of NADH-Dependent Glutamate Synthase Protein and in the Activity of the Enzyme in Roots of Rice Seedlings

When rice seedlings, after the growth for 26 days in water alone,were transferred to nutrient medium contained 1 mM NH₄Cl, thelevel of NADH-dependent glutamate synthase (GOGAT) protein andthe activity of the enzyme increased more than 10-fold in root,but not in shoots. Both the level of the protein and the activityreached a maximum within 24 h. NH₄Cl was effective at concentrationsas low as 50 µM. A supply of either 1 mM NaNO₃ or 0.5mM NH₄NO₃ also caused such increases, but NHa₄Cl was most effective.A supply of glutamine or glutamate was less effective. The increasewas specific to NADH-GOGAT and little change was observed inthe levels of ferredoxin-GOGAT and glutamine synthetase isoproteinsin roots. These inducible increases in the levels of NADH-GOGATprotein and in its activity were greater in the root-tip regionthan at the base of the root. Both 6-methylpurine and cycloheximidecompletely inhibited the effects of NH₄Cl. Moreover, the mRNAfor NADH-GOGAT in rice roots accumulated markedly within 12h of the start of a supply of NH₄Cl. A possible role for therapid response of NADH-GOGAT to a supply of NH₄C1 is discussed. (Received May 18, 1995; Accepted July 10, 1995)     

Gene therapy for sickle cell disease: An update

Sickle cell disease (SCD) is one of the most common life-threatening monogenic diseases affecting millions of people worldwide. Allogenic hematopietic stem cell transplantation is the only known cure for the disease with high success rates, but the limited availability of matched sibling donors and the high risk of transplantation-related side effects force the scientific community to envision additional therapies. Ex vivo gene therapy through globin gene addition has been investigated extensively and is currently being tested in clinical trials that have begun reporting encouraging data. Recent improvements in our understanding of the molecular pathways controlling mammalian erythropoiesis and globin switching offer new and exciting therapeutic options. Rapid and substantial advances in genome engineering tools, particularly CRISPR/Cas9, have raised the possibility of genetic correction in induced pluripotent stem cells as well as patient-derived hematopoietic stem and progenitor cells. However, these techniques are still in their infancy, and safety/efficacy issues remain that must be addressed before translating these promising techniques into clinical practice.     

Development of a simple assay system for protein-stabilizing efficiency based on hemoglobin protection against denaturation and measurement of the cooperative effect of mixing protein stabilizers

We have elucidated the cooperative stabilization of proteins by sugars, amino acids, and other protein-stabilizing agents using a new and simple assay system. Our system determines the protein-stabilizing ability of various compounds by measuring their ability to protect hemoglobin from denaturation. Hemoglobin denaturation was readily measured by quantitative changes in its ultraviolet–visible absorption spectrum. The efficiency of our assay was confirmed using various sugars such as trehalose and sucrose that are known to be good protein stabilizers. We have also found that mixtures of two different types of protein stabilizers resulted in a cooperative stabilizing effect on protein.