A Method for Assaying Deubiquitinating Enzymes

A general method for the assay of deubiquitinating enzymes was described in detail using (125)I-labeled ubiquitin-fused αNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products. Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells

UV to near-IR observations of the DART-Dimorphos collision

The impact of the Double Asteroid Redirection Test (DART) spacecraft with Dimorphos allows us to study asteroid collision physics, including momentum transfer, the ejecta properties, and the visibility of such events in the Solar System. We report observations of the DART impact in the ultraviolet (UV), visible light, and near-infrared (IR) wavelengths. The observations support the existence of at least two separate components of the ejecta: a fast and a slow component. The fast-ejecta component is composed of a gaseous phase, moving at about 1.6 km/s with a mass of <10^4 kg. The fast ejecta is detected in the UV and visible light, but not in the near-IR $z$-band observations. Fitting a simplified optical thickness model to these observations allows us to constrain some of the properties of the fast ejecta, including its scattering efficiency and the opacity of the gas. The slow ejecta component is moving at typical velocities of up to about 10 m/s. It is composed of micrometer-size particles, that have a scattering efficiency, at the direction of the observer, of the order of 10^-3 and a total mass of about 10^6 kg. The larger particles in the slow ejecta, whose size is bound to be in the range between ~1 mm to ~1 m, likely have a scattering efficiency larger than that of the pre-impact Didymos system.Comment: Submitted to MNRA

SOXS: a wide band spectrograph to follow up transients

SOXS (Son Of X-Shooter) will be a spectrograph for the ESO NTT telescope capable to cover the optical and NIR bands, based on the heritage of the X-Shooter at the ESO-VLT. SOXS will be built and run by an international consortium, carrying out rapid and longer term Target of Opportunity requests on a variety of astronomical objects. SOXS will observe all kind of transient and variable sources from different surveys. These will be a mixture of fast alerts (e.g. gamma-ray bursts, gravitational waves, neutrino events), mid-term alerts (e.g. supernovae, X-ray transients), fixed time events (e.g. close-by passage of minor bodies). While the focus is on transients and variables, still there is a wide range of other astrophysical targets and science topics that will benefit from SOXS. The design foresees a spectrograph with a Resolution-Slit product ~ 4500, capable of simultaneously observing over the entire band the complete spectral range from the U- to the H-band. The limiting magnitude of R~20 (1 hr at S/N~10) is suited to study transients identified from on-going imaging surveys. Light imaging capabilities in the optical band (grizy) are also envisaged to allow for multi-band photometry of the faintest transients. This paper outlines the status of the project, now in Final Design Phase.Comment: 12 pages, 14 figures, to be published in SPIE Proceedings 1070

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer

The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells

INTRODUCTION: Loss of the cyclin-dependent kinase inhibitor p27 is associated with poor prognosis in breast cancer. The decrease in p27 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S phase kinase protein 2 (Skp2). The mammalian target of rapamycin (mTOR) is a downstream mediator in the phosphoinositol 3' kinase (PI3K)/Akt pathway that down-regulates p27 levels in breast cancer. Rapamycin was found to stabilize p27 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression is unknown. METHODS: The expression of Skp2 mRNA and protein levels were examined in rapamycin-treated breast cancer cell lines. The effect of rapamycin on the degradation rate of Skp2 expression was examined in cycloheximide-treated cells and in relationship to the anaphase promoting complex/Cdh1 (APC\C) inhibitor Emi1. RESULTS: Rapamycin significantly decreased Skp2 mRNA and protein levels in a dose and time-dependent fashion, depending on the sensitivity of the cell line to rapamycin. The decrease in Skp2 levels in the different cell lines was followed by cell growth arrest at G1. In addition, rapamycin enhanced the degradation rate of Skp2 and down-regulated the expression of the APC\C inhibitor Emi1. CONCLUSION: These results suggest that Skp2, an important oncogene in the development and progression of breast cancer, may be a novel target for rapamycin treatment

Acute renal failure following deferoxamine overdose

A 17-year-old patient with sickle cell-beta thalassemia undergoing treatment with home iron chelation therapy inadvertently received ten times the recommended dose of intravenous deferoxamine. Acute renal failure (ARF) developed within hours. Immediate treatment with high-efficiency hemodialysis resulted in the prompt return of renal function after only one hemodialysis session. No long-term nephrotoxic effects of the deferoxamine overdose developed after more than 1 year of follow-up. Children with sickle cell disease who are on intravenous deferoxamine and their parents should be cautioned about the possibility of ARF with overdose due to malfunction of the pump and/or inadequate monitoring during treatment. ARF, should it occur in such children, appears to respond well to treatment with high-efficiency hemodialysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42309/1/s00467-002-1051-7.pd