Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19–469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

Effect of Subacute Intoxication with Nitrite on AMP-Deaminase Activity in Skeletal Muscles of Common Carp (Cyprinus carpio)

This study was aimed to evaluate the effects of intoxication of common carp with nitrite on some properties of Adenosine 5´-monophosphate (AMP) deaminase in the skeletal muscle. LC50 value was determined to be 512 mg/L. After estimating the LC50, fish were exposed to 153 mg/L (30% LC50) and sampling was done at 0, 24, 48, 72, and 96 h after exposure. Cytosolic AMP deaminase was purified from the skeletal muscle of the control and experimental groups. AMP-deaminase showed Hill kinetics with an S0.5 value for AMP of 2.52±0.031 and 1.45±0.07 mM, a Hill coefficient (nH) of 1.35±0.051 and 1.79±0.60, and a maximal velocity (Vmax) of 133.39±28 and 98.9±38 IU/mg protein for the control and experimental groups, respectively after 96 h. The optimum pH was reduced during nitrite intoxication. The maximal activation of the enzyme was at 45ºC. Also, activation energy declined in the experimental groups. Fenton reaction showed that the enzyme was tolerant to free radicals. According to the results, AMP deaminase activity in the common carp’s muscles increased following nitrite intoxication. Also, acidic condition increased AMP deaminase activity that could be an adaptive response to hypoxic condition caused by nitrite intoxication.

CONTROLLING THE CELL CYCLE RESTRICTION SWITCH ACROSS THE INFORMATION GRADIENT

Boolean models represent a drastic simplification of complex biomolecular systems, and yet accurately predict system properties, e.g., effective control strategies. Why is this? Parameter robustness has been highlighted as a general feature of biomolecular systems and may play an important role in the accuracy of Boolean models. We argue here that a useful way to view a system’s controllability properties is through its repertoire of self-sustaining positive circuits (stable motifs). We examine attractor control and self-sustaining circuits within the cell cycle restriction switch, a bistable regulatory circuit that allows or prevents entry into the cell cycle. We explore this system using three models: a previously published Boolean model, a Hill kinetics model that we construct from the Boolean model using the HillCube methodology, and a reaction-based model we construct from the literature. We highlight the robustness of stable motifs across these three levels of modeling detail. We also show how consideration of control-robust regulatory circuits can aid in parameter specification.

Hill kinetics as a noise filter: the role of transcription factor autoregulation in gene cascades

Multiple genes under control of a single, self-regulating regulator can exhibit different (binary or graded) responses to the same signal.