A Convenient, Rapid, Sensitive, and Reliable Spectrophotometric Assay for Adenylate Kinase Activity.

Enzymatic activity assays are essential and critical for the study of enzyme kinetics. Adenylate kinase (Adk) plays a fundamental role in cellular energy and nucleotide homeostasis. To date, assays based on different principles have been used for the determination of Adk activity. Here, we show a spectrophotometric analysis technique to determine Adk activity with bromothymol blue as a pH indicator. We analyzed the effects of substrates and the pH indicator on the assay using orthogonal design and then established the most optimal assay for Adk activity. Subsequently, we evaluated the thermostability of Adk and the inhibitory effect of KCl on Adk activity with this assay. Our results show that this assay is simple, rapid, and precise. It shows great potential as an alternative to the conventional Adk activity assay. Our results also suggest that orthogonal design is an effective approach, which is very suitable for the optimization of complex enzyme reaction conditions.

Cross-talk between juvenile hormone and ecdysone regulates transcription of fibroin modulator binding protein-1 in Bombyx mori.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are the most important hormones in silkworm and play vital roles in silkworm development, metamorphosis, and silk protein synthesis. Fibroin modulator binding protein-1 (FMBP-1) is a novel transcription factor regulating fibroin heavy chain (fib-H) transcription in Bombyx mori. The roles of JH and 20E on FMBP-1 transcription are less known. Here, we show FMBP-1 transcription is repressed by juvenile hormone analog (JHA) and activated by 20E. We identify two Krüppel homolog 1 (Kr-h1) binding sites (KBS1 and KBS2) and an E74A binding site (EBS) in the promoter of FMBP-1. We demonstrate Kr-h1 directly binds to KBS1 and KBS2 to repress FMBP-1 transcription, and 20E promotes FMBP-1 transcription through E74A. In the presence of JH and 20E, E74A abolishes the repression of Kr-h1 and activates FMBP-1 transcription through direct binding to EBS between KBS1 and KBS2 in FMBP-1 promoter. Further, JHA and 20E treatment and RNA interference confirm the effects of JH and 20E on FMBP-1 transcription in vivo, thus affecting fib-H transcription. Our results reveal the molecular mechanism of FMBP-1 transcription regulated by the cross-talk between JH and 20E in Bombyx mori, and provide novel insights into FMBP-1 transcriptional regulation and silk protein synthesis.

In Situ Synthesis of Silver Nanoparticles on the Polyelectrolyte-Coated Sericin/PVA Film for Enhanced Antibacterial Application.

To develop silk sericin (SS) as a potential antibacterial biomaterial, a novel composite of polyelectrolyte multilayers (PEMs) coated sericin/poly(vinyl alcohol) (SS/PVA) film modified with silver nanoparticles (AgNPs) has been developed using a layer-by-layer assembly technique and ultraviolet-assisted AgNPs synthesis method. Ag ions were enriched by PEMs via the electrostatic attraction between Ag ions and PEMs, and then reduced to AgNPs in situ with the assistance of ultraviolet irradiation. PEMs facilitated the high-density growth of AgNPs and protected the synthesized AgNPs due to the formation of a 3D matrix, and thus endowed SS/PVA film with highly effective and durable antibacterial activity. Scanning electron microscopy, energy dispersive spectroscopy, X-ray diffractometry, Fourier transfer infrared spectroscopy, water contact angle, mechanical property and thermogravimetric analysis were applied to characterize SS/PVA, PEMs-SS/PVA and AgNPs-PEMs-SS/PVA films, respectively. AgNPs-PEMs-SS/PVA film has exhibited good mechanical performance, hydrophilicity, water absorption capability as well as excellent and durable antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and good stability and degradability. This study has developed a simple method to design and prepare AgNPs-PEMs-SS/PVA film for potential antibacterial application.