Phosphorylation of MCPH1 isoforms during mitosis followed by isoform-specific degradation by APC/C-CDH1.

Microcephalin-1 (MCPH1) exists as 2 isoforms that regulate cyclin-dependent kinase-1 activation and chromosome condensation during mitosis, with MCPH1 mutations causing primary microcephaly. MCPH1 is also a tumor suppressor protein, with roles in DNA damage repair/checkpoints. Despite these important roles, there is little information on the cellular regulation of MCPH1. We show that both MCPH1 isoforms are phosphorylated in a cyclin-dependent kinase-1-dependent manner in mitosis and identify several novel phosphorylation sites. Upon mitotic exit, MCPH1 isoforms were degraded by the anaphase-promoting complex/cyclosome-CDH1 E3 ligase complex. Anaphase-promoting complex/cyclosome-CDH1 target proteins generally have D-Box or KEN-Box degron sequences. We found that MCPH1 isoforms are degraded independently, with the long isoform degradation being D-Box dependent, whereas the short isoform was KEN-Box dependent. Our research identifies several novel mechanisms regulating MCPH1 and also highlights important issues with several commercial MCPH1 antibodies, with potential relevance to previously published data.-Meyer, S. K., Dunn, M., Vidler, D. S., Porter, A., Blain, P. G., Jowsey, P. A. Phosphorylation of MCPH1 isoforms during mitosis followed by isoform-specific degradation by APC/C-CDH1.

Noninvasive in vivo magnetic resonance measures of glutathione synthesis in human and rat liver as an oxidative stress biomarker.

UNLABELLED: Oxidative stress (OS) plays a central role in the progression of liver disease and in damage to liver by toxic xenobiotics. We have developed methods for noninvasive assessment of hepatic OS defenses by measuring flux through the glutathione (GSH) synthesis pathway. (13) C-labeled GSH is endogenously produced and detected by in vivo magnetic resonance after administration of [2-(13) C]-glycine. We report on a successful first-ever human demonstration of this approach as well as preclinical studies demonstrating perturbed GSH metabolism in models of acute and chronic OS. Human studies employed oral administration of [2-(13) C]-glycine and (13) C spectroscopy on a 3T clinical magnetic resonance (MR) imaging scanner and demonstrated detection and quantification of endogenously produced (13) C-GSH after labeled glycine ingestion. Plasma analysis demonstrated that glycine (13) C fractional enrichment achieved steady state during the 6-hour ingestion period. Mean rate of synthesis of hepatic (13) C-labeled GSH was 0.32 ± 0.18 mmole/kg/hour. Preclinical models of acute OS and nonalcoholic steatohepatitis (NASH) comprised CCl4 -treated and high-fat, high-carbohydrate diet-fed Sprague-Dawley rats, respectively, using intravenous administration of [2-(13) C]-glycine and observation of (13) C-label metabolism on a 7T preclinical MR system. Preclinical studies demonstrated a 54% elevation of GSH content and a 31% increase in flux through the GSH synthesis pathway at 12 hours after acute insult caused by CCl4 administration, as well as a 23% decrease in GSH content and evidence of early steatohepatitis in the model of NASH. CONCLUSION: Our data demonstrate in vivo (13) C-labeling and detection of GSH as a biomarker of tissue OS defenses, detecting chronic and acute OS insults. The methods are applicable to clinical research studies of hepatic OS in disease states over time as well as monitoring effects of therapeutic interventions.

Rapid and complete bioavailability of antidotes for organophosphorus nerve agent and cyanide poisoning in minipigs after intraosseous administration.

STUDY OBJECTIVE: Management of chemical weapon casualties includes the timely administration of antidotes without contamination of rescuers. Personal protective equipment makes intravenous access difficult but does not prevent intraosseous drug administration. We therefore measured the systemic bioavailability of antidotes for organophosphorus nerve agent and cyanide poisoning when administered by the intraosseous, intravenous, and intramuscular routes in a small study of Göttingen minipigs. METHODS: Animals were randomly allocated to sequentially receive atropine (0.12 mg/kg by rapid injection), pralidoxime (25 mg/kg by injection during 2 minutes), and hydroxocobalamin (75 mg/kg during 10 minutes) by the intravenous or intraosseous route, or atropine and pralidoxime by the intramuscular route. Plasma concentrations were measured for 6 hours to characterize the antidote concentration-time profiles for each route. RESULTS: Maximum plasma concentrations of atropine and pralidoxime occurred within 2 minutes when administered by the intraosseous route compared with 8 minutes by the intramuscular route. Maximum plasma hydroxocobalamin concentration occurred at the end of the infusion when administered by the intraosseous route. The mean area under the concentration-time curve by the intraosseous route was similar to the intravenous route for all 3 drugs and similar to the intramuscular route for atropine and pralidoxime. CONCLUSION: This study showed rapid and substantial antidote bioavailability after intraosseous administration that appeared similar to that of the intravenous route. The intraosseous route of antidote administration should be considered when intravenous access is difficult.

Analysis of organometal(loid) compounds in environmental and biological samples.

Measurement of the different physicochemical forms of metals and metalloids is a necessary pre-requisite for the detailed understanding of an element's interaction with environmental and biological systems. Such chemical speciation data is important in a range of areas, including toxicology, ecotoxicology, biogeochemistry, food safety and nutrition. This chapter considers developments in the speciation analysis of organometallic compounds (OMCs), focusing on those of As, Hg, Se and Sn. Typically, organometallic analysis requires a chromatographic separation prior to analyte detection and gas chromatography (GC), high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) can serve this purpose. Following separation, detection is achieved using element specific detectors (ESDs) such as inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma optical emission spectroscopy (ICP-OES), atomic fluorescence spectrometry (AFS), atomic absorption spectrometry (AAS) or atmospheric pressure ionization mass spectrometry (API-MS). Techniques employing a vapor generation (VG) stage prior to detection are also discussed. Complementary structural and quantitative data may be acquired through the combination of elemental and molecular mass spectrometry. The advantages and disadvantages of the various analytical systems are discussed, together with issues related to quantification and quality management.

Potential for using isotopically altered metalloproteins in species-specific isotope dilution analysis of proteins by HPLC coupled to inductively coupled plasma mass spectrometry.

The production and evaluation of an isotopically enriched metalloprotein standard for use as a calibrant in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is described. Using a model system involving the copper-containing protein rusticyanin (Rc) from the bacterium Acido-thiobacillus ferrooxidans, it was possible to demonstrate the analytical conditions that could be used for the measurement of metalloproteins by on-line IDMS analysis. Rc was chosen because it is a well-characterized protein with an established amino acid sequence and can be produced in suitable quantities using a bacterial recombinant system. Three different forms of the protein were studied by organic and inorganic mass spectrometry: the native form of the protein containing a natural isotopic profile for copper, an isotopically enriched species containing virtually all of its copper as the 65Cu isotope, and the nonmetalated apo form. Incorporation of the copper isotopes into the apo form of the protein was determined using a UV-vis spectrophotometric assay and shown to be complete for each of the copper-containing species. The experimental conditions required to maintain the conformational form of the protein with a nonexchangeable copper center were established using +ve electrospray mass spectrometry. A pH 7.0 buffer was found to afford the most appropriate conditions, and this was then used with HPLC-ICP-MS to verify the stability of the copper center by analysis of mixtures of different isotopic solutions. No exchange of the enriched copper isotope from Rc with an added naturally abundant inorganic copper cation was observed under a neutral pH environment, indicating that species-specific ID-MS analysis of metalloproteins is possible.