TIMP-3 inhibition of ADAMTS-4 (Aggrecanase-1) is modulated by interactions between aggrecan and the C-terminal domain of ADAMTS-4

ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K(m) in the 10 microm range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.     

Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

Methods development for diffraction and spectroscopy studies of metalloenzymes at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) open up new possibilities for X-ray crystallographic and spectroscopic studies of radiation-sensitive biological samples under close to physiological conditions. To facilitate these new X-ray sources, tailored experimental methods and data-processing protocols have to be developed. The highly radiation-sensitive photosystem II (PSII) protein complex is a prime target for XFEL experiments aiming to study the mechanism of light-induced water oxidation taking place at a Mn cluster in this complex. We developed a set of tools for the study of PSII at XFELs, including a new liquid jet based on electrofocusing, an energy dispersive von Hamos X-ray emission spectrometer for the hard X-ray range and a high-throughput soft X-ray spectrometer based on a reflection zone plate. While our immediate focus is on PSII, the methods we describe here are applicable to a wide range of metalloenzymes. These experimental developments were complemented by a new software suite, cctbx.xfel. This software suite allows for near-real-time monitoring of the experimental parameters and detector signals and the detailed analysis of the diffraction and spectroscopy data collected by us at the Linac Coherent Light Source, taking into account the specific characteristics of data measured at an XFEL.     

Selection of a Streptomyces strain able to produce cell wall degrading enzymes and active against Sclerotinia sclerotiorum

Control of plant pathogen Sclerotinia sclerotiorum is an ongoing challenge because of its wide host range and the persistence of its sclerotia in soil. Fungicides are the most commonly used method to control this fungus but these can have ecotoxicity impacts. Chitinolytic Streptomyces strains isolated from Brazilian tropical soils were capable of inhibiting S. sclerotiorum growth in vitro, offering new possibilities for integrated pest management and biocontrol, with a new approach to dealing with an old problem. Strain Streptomyces sp. 80 was capable of irreversibly inhibiting fungal growth. Compared to other strains, its crude enzymes had the highest chitinolytic levels when measured at 25°C and strongly inhibited sclerotia from S. sclerotiorum. It produced four hydrolytic enzymes involved in fungal cell wall degradation when cultured in presence of the fungal mycelium. The best production, obtained after three days, was 0.75 U/ml for exochitinase, 0.9 U/ml for endochitinase, 0.16 U/ml for glucanase, and 1.78 U/ml for peptidase. Zymogram analysis confirmed two hydrolytic bands of chitinolytic activity with apparent molecular masses of 45.8 and 206.8 kDa. One glucanase activity with an apparent molecular mass of 55 kDa was also recorded, as well as seven bands of peptidase activity with apparent molecular masses ranging from 15.5 to 108.4 kDa. Differential interference contrast microscopy also showed alterations of hyphal morphology after co-culture. Streptomyces sp. 80 seems to be promising as a biocontrol agent against S. sclerotiorum, contributing to the development of new methods for controlling plant diseases and reducing the negative impact of using fungicides.     

Bolus administration of obestatin does not change glucose and insulin levels neither in the systemic nor in the portal circulation of the rat

Obestatin is a second peptide derived from the preproghrelin polypeptide. It was originally thought to have anorexigenic effects, thereby functioning as an antagonist of ghrelin. However, this has been a subject of debate ever since. Since acylated ghrelin strongly induces insulin resistance, it could be hypothesized that obestatin plays a role in glucose homeostasis as well. In the present study we evaluated the effect of obestatin on glucose and insulin metabolism in the systemic and portal circulation. Obestatin 200 nmol/kg was administered systemically as a single intravenous bolus injection to fasted pentobarbital anesthetized adult male Wistar rats. Up to 50 min after administration, blood samples were taken to measure glucose and insulin concentrations, both in the portal and in the systemic circulation. The effect of obestatin was evaluated in fasted and in glucose-stimulated conditions (IVGTT) and compared to control groups treated with saline or IVGTT, respectively. Intravenous administration of obestatin did not have any effect on glucose and insulin concentrations, neither systemic nor portal, when compared to the control groups. Only the glucose peak 1 min after administration of IVGTT was slightly higher in the obestatin treated rats: 605.8 ± 106.3% vs. 522.2 ± 47.1% in the portal circulation, respectively (NS), and 800.7 ± 78.7% vs. 549.6 ± 37.0% in the systemic circulation, respectively (P < 0.02), but it can be debated whether this has any clinical relevance. In the present study, we demonstrated that intravenously administered obestatin does not influence glucose and insulin concentrations, neither in the portal nor in the systemic circulation.     

Neural and cardiovascular responses to emotional stress in humans

Sympathetic neural responses to mental stress are well documented but controversial, whereas sympathetic neural responses to emotional stress are unknown. The purpose of this study was to investigate neural and cardiovascular responses to emotional stress evoked by negative pictures and reexamine the relationship between muscle sympathetic nerve activity (MSNA) and perceived stress. Mean arterial pressure (MAP), heart rate (HR), MSNA, and perceived stress levels were recorded in 18 men during three randomized trials: 1) neutral pictures, 2) negative pictures, and 3) mental stress. MAP and HR increased during mental stress (Delta14 +/- 2 mmHg and Delta15 +/- 2 beats/min, P < 0.001) but did not change during viewing of negative or neutral pictures. MSNA did not change during viewing of neutral (Delta1 +/- 1 burst/min, n = 16) or negative (Delta0 +/- 1 burst/min, n = 16) pictures or during mental stress (Delta1 +/- 2 burst/min, n = 13). Perceived stress levels were higher during mental stress (3 +/- 0 arbitrary units) than during viewing negative pictures (2 +/- 0 arbitrary units, P < 0.001). Perceived stress levels were not correlated to changes in MSNA during negative pictures (r = 0.10, P = 0.84) or mental stress (r = 0.36, P = 0.23). In conclusion, our results demonstrate robust increases in MAP and HR during mental stress, but not during emotional stress evoked by negative pictures. Although the influence of mental stress on MSNA remains unresolved, our findings challenge the concept that perceived stress levels modulate MSNA during mental stress.