Quantitative Förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet–SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k_cat, K_M, and catalytic efficiency (k_cat/K_M) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.     

Near-infrared time-resolved optical absorption studies of the reaction of fully reduced cytochrome c oxidase with dioxygen

Electron transfer during the reaction of fully reduced bovine heart cytochrome oxidase with dioxygen has been studied at 24 degrees C in the near-infrared region following photolysis of the fully reduced CO-bound complex. The transient spectral changes and kinetics were followed on microsecond to millisecond time scales at nine different wavelengths between 597 and 935 nm and were analyzed using singular value decomposition and global exponential fitting. Four apparent lifetimes, 14 micros, 40 micros, 86 micros, and 1.1 ms, were resolved. The near-infrared spectra of the intermediates are extracted on the basis of a previously proposed mechanism [Sucheta et al. (1998) Biochemistry 37, 17905-17914] and compared to model spectra of the postulated intermediates. The data provide a comprehensive picture of the spectral contributions of the different redox centers in their respective oxidation or ligation states in the near-infrared region and strongly support that Cu(A) is partially (2/3), but not fully, oxidized in the 3-electron-reduced ferryl intermediate.     

Taurine transporter is expressed in vascular smooth muscle cells

Summary. The regulation of vascular smooth muscle cells (VSMCs) function by taurine has been a subject of increasing interest and investigation, and taurine is taken up into cells through a specific transporter system, the taurine transporter (TAUT). In the present study, we examined the expression of TAUT in VSMCs and the kinetic parameters of the uptake process of TAUT in VSMCs. RT-PCR and western blot demonstrated that the mRNA and protein of TAUT was expressed in VSMCs in vitro. Immunohistochemistry using antibody for TAUT revealed the expression of this protein in rat thoracic aorta. The maximal [³H]taurine uptake rate in VSMCs was 37.75 ± 3.13 pmol/min per mg of protein, with a K _m value of 5.42 ± 0.81 μM. Thus, VSMCs are able to express a functional taurine transporter. The regulation and detailed function of taurine and TAUT in VSMCs remain unclear, but our findings suggest a functional role for them in VSMCs metabolism.     

HIV/AIDS in Asia： The Shape of Epidemics and Their Molecular Epidemiology

The Asia-Pacific region is a home to 60% of the population in the world and to approximately one quarter of people with HIV/AIDS. Close to a million of people has been infected and a half million people died of AIDS annually in Asia,becoming the second largest epicenter of global AIDS epidemic. Molecular epidemiology has been useful tool to track a course of HIV spread. In-depth knowledge from the studies on molecular epidemiology elucidates the dynamics of HIV spread and the interrelationship of epidemics in the different regions in Asia.     

Indoor Thin‐Film Photovoltaics: Progress and Challenges

Energy generation and consumption have always been an important component of social development. Interests in this field are beginning to shift to indoor photovoltaics (IPV) which can serve as power sources under low light conditions to meet the energy needs of rapidly growing fields, such as intelligence gathering and information processing which usually operate via the Internet‐of‐things (IoT). Since the power requirements for this purpose continue to decrease, IPV systems under low light may facilitate the realization of self‐powered high‐tech electronic devices connected through the IoT. This review discusses and compares the characteristics of different types of IPV devices such as those based on silicon, dye, III‐V semiconductors, organic compounds, and halide perovskites. Among them, specific attention is paid to perovskite photovoltaics which may potentially become a high performing IPV system due to the fascinating photophysics of the halide perovskite active layer. The limitations of such indoor application as they relate to the toxicity, stability, and electronic structure of halide perovskites are also discussed. Finally, strategies which could produce highly functional, nontoxic, and stable perovskite photovoltaics devices for indoor applications are proposed.     

Microbial diversity in deep-sea sediment from the cobalt-rich crust deposit region in the Pacific Ocean

Cobalt-rich crusts are important metallic mineral resources with great economic potential, usually distributed on seamounts located in the Pacific Ocean. Microorganisms are believed to play a role in the formation of crusts as well as in metal cycling. To explore the microbial diversity related to cobalt-rich crusts, 16S ribosomal RNA gene clone libraries were constructed from three consecutive sediment layers. In total, 417 bacterial clones were obtained from three bacterial clone libraries, representing 17 distinct phylogenetic groups. Proteobacteria dominated in the bacterial communities, followed by Acidobacteria and Planctomycetes. Compared with high bacterial diversity, archaea showed a remarkably low diversity, with all 137 clones belonging to marine archaeal group I except one novel euryarchaeotal clone. The microbial communities were potentially involved in sulfur, nitrogen and metal cycling in the area of cobalt-rich crusts. Sulfur oxidation and metal oxidation were potentially major sources of energy for this ecosystem. This is the first reported investigation of microbial diversity in sediments associated with cobalt-rich crusts, and it casts fresh light on the microbial ecology of these important ecosystems.