Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon

Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis matures from Pro-Tk-subtilisin (Pro-TKS) upon autoprocessing and degradation of propeptide. Pro-TKS contains the insertion sequence (IS1) at the N-terminus of the mature domain as compared to bacterial pro-subtilisins. To analyze the role of IS1, the Pro-TKS derivative without IS1 (?IS1-Pro-TKS) and its active-site mutants (?IS1-Pro-S324A and ?IS1-Pro-S324C) were constructed and characterized. ?IS1-Pro-S324A and ?IS1-Pro-TKS represent an unautoprocessed and autoprocessed form of ?IS1-Pro-TKS, respectively. The CD and ANS fluorescence spectra of these proteins indicate that folding of ?IS1-Pro-TKS is not completed by binding of Ca²⁺ ions but is completed by the subsequent autoprocessing reaction. Thermal denaturation of these proteins analyzed by DSC and CD spectroscopy indicates that unautoprocessed ?IS1-Pro-TKS is less stable than autoprocessed ?IS1-Pro-TKS by 26.3?°C in T _m. The stability of autoprocessed ?IS1-Pro-TKS is comparable to that of Pro-TKS, which is slightly lower than that of unautoprocessed Pro-TKS. These results suggest that ?IS1-Pro-TKS is fully folded and greatly stabilized by autoprocessing. ?IS1-Pro-TKS more slowly matured to ?IS1-Tk-subtilisin than Pro-TKS did, due to a decrease in the autoprocessing rate. We propose that IS1 is required not only for hyperstabilization of Pro-TKS but also for its rapid maturation.     

Cell movements of the deep layer of non-neural ectoderm underlie complete neural tube closure in Xenopus

In developing vertebrates, the neural tube forms from a sheet of neural ectoderm by complex cell movements and morphogenesis. Convergent extension movements and the apical constriction along with apical-basal elongation of cells in the neural ectoderm are thought to be essential for the neural tube closure (NTC) process. In addition, it is known that non-neural ectoderm also plays a crucial role in this process, as the neural tube fails to close in the absence of this tissue in chick and axolotl. However, the cellular and molecular mechanisms by which it functions in NTC are as yet unclear. We demonstrate here that the non-neural superficial epithelium moves in the direction of tensile forces applied along the dorsal-ventral axis during NTC. We found that this force is partly attributable to the deep layer of non-neural ectoderm cells, which moved collectively towards the dorsal midline along with the superficial layer. Moreover, inhibition of this movement by deleting integrin β1 function resulted in incomplete NTC. Furthermore, we demonstrated that other proposed mechanisms, such as oriented cell division, cell rearrangement and cell-shape changes have no or only minor roles in the non-neural movement. This study is the first to demonstrate dorsally oriented deep-cell migration in non-neural ectoderm, and suggests that a global reorganization of embryo tissues is involved in NTC.     

Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.     

Decrease of Peripheral and Intestinal NKG2A-Positive T Cells in Patients with Ulcerative Colitis

To investigate the role of inhibitory natural killer receptors (iNKRs) in inflammatory bowel disease (IBD), we analyzed the expression of NKG2A, one of the iNKRs, on T cells in a mouse colitis model and human IBD. During the active phase of dextran sulfate sodium (DSS)-induced mouse colitis, the frequency of NKG2A+ T cells was significantly decreased in the peripheral blood, and increased in the intestine, suggesting the mobilization of this T cell subset to the sites of inflammation. Administration of anti-NKG2A antibody increased the number of inflammatory foci in DSS-induced colitis, suggesting the involvement of NKG2A+ T cells in this colitis model. In ulcerative colitis (UC) patients, the frequency of peripheral blood NKG2A+ T cells was significantly decreased, compared with Crohn's disease (CD) patients and healthy controls, regardless of clinical conditions such as treatment modalities and disease activity. Notably, in sharp contrast to the DSS-induced mouse colitis model, the frequency of NKG2A+ cells among intestinal T cells was also decreased in UC patients. These results suggest that inadequate local infiltration of NKG2A+ T cells may be involved in the pathogenesis of UC.     

High-Speed Imaging of Amoeboid Movements Using Light-Sheet Microscopy

Light-sheet microscopy has been developed as a powerful tool for live imaging in biological studies. The efficient illumination of specimens using light-sheet microscopy makes it highly amenable to high-speed imaging. We therefore applied this technology to the observation of amoeboid movements, which are too rapid to capture with conventional microscopy. To simplify the setup of the optical system, we utilized the illumination optics from a conventional confocal laser scanning microscope. Using this set-up we achieved high-speed imaging of amoeboid movements. Three-dimensional images were captured at the recording rate of 40 frames/s and clearly outlined the fine structures of fluorescent-labeled amoeboid cellular membranes. The quality of images obtained by our system was sufficient for subsequent quantitative analysis for dynamics of amoeboid movements. This study demonstrates the application of light-sheet microscopy for high-speed imaging of biological specimens.     

Backbone chemical shifts assignments, secondary structure, and ligand binding of a family GH-19 chitinase from moss, Bryum coronatum

Family GH19 chitinases have been recognized as important in the plant defense against fungal pathogens. However, their substrate-recognition mechanism is still unknown. We report here the first resonance assignment of NMR spectrum of a GH19 chitinase from moss, Bryum coronatum (BcChi-A). The backbone signals were nearly completely assigned, and the secondary structure was estimated based on the chemical shift values. The addition of the chitin dimer to the enzyme solution perturbed the chemical shifts of HSQC resonances of the amino acid residues forming the putative substrate-binding cleft. Further NMR analysis of the ligand binding to BcChi-A will improve understanding of the substrate-recognition mechanism of GH-19 enzymes.     

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis

Xenotropic murine leukemia virus-related virus (XMRV) is a virus generated under artificial conditions by the recombination of 2 murine leukemia virus (MLV) proviruses, PreXMRV-1 and PreXMRV-2, during the in vivo passage of human prostate cancer cells in athymic nude mice. The molecular etiology of XMRV infection has not been characterized and its implication in human prostate cancer progression remains equivocal. As a step toward resolving this issue we developed an in vitro enzymatic assay system to characterize XMRV protease (PR)-mediated cleavage of host-cell proteins. Enzymatically-active XMRV PR protein was synthesized using a wheat-germ cell-free system. By monitoring cleavage activity of XMRV PR by AlphaScreen and 2-color immunoblot analyses, we revealed that the catalytic activity of XMRV PR is selectively blocked by the HIV PR inhibitor, Amprenavir, and identified several human tumor suppressor proteins, including PTEN and BAX, to be substrates of XMRV PR. This system may provide an attractive means for analyzing the function of retrovirus proteases and provide a technology platform for drug screening.     

Cyclic dipeptides exhibit potency for scavenging radicals

Twenty kinds of cyclic dipeptides containing l-leucine were synthesized, and their antioxidant activity against ·OH and O(2)(·-) was investigated. Compounds possessing polar amino acid residues, such as Asp, Cys, Glu, Lys, Pro, Ser, and Trp, exhibited higher antioxidant activity against ·OH than vitamin E. However, only cyclo(l-Cys-l-Leu) scavenged O(2)(·-).