Spatial and temporal effects in protein post-translational modification distributions in the developing mouse brain.

Protein post-translational modification (PTM) is a powerful way to modify the behavior of cellular proteins and thereby cellular behavior. Multiple recent studies of evolutionary trends have shown that certain pairs of protein post-translational modifications tend to occur closer to each other than expected at random. This type of observation may form the basis of a proposed "PTM code", whereby protein function is controlled by complex patterns of multiple PTMs. This code could provide an additional, powerful level of regulatory control for protein function and is a plausible explanation for observations of increasingly frequent and diverse protein modification in cell biology. In this study, we use mass spectrometry and proteomic strategies to present biological data showing spatiotemporal PTM co-localization across multiple PTM categories, which display changes over development of the brain. This may be an indication of the existence of a PTM-based functional coding mechanism, which would significantly expand our view of the ways in which cells use protein PTMs in complex signaling networks.

The acyl-CoA binding protein is required for normal epidermal barrier function in mice.

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ∼50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.

A novel method for the simultaneous enrichment, identification, and quantification of phosphopeptides and sialylated glycopeptides applied to a temporal profile of mouse brain development.

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.

Cerebral state monitoring in Beagle dogs sedated with medetomidine.

OBJECTIVE: To provide experience of monitoring the level of hypnosis with the Cerebral State Monitor (CSM), a device extracting a single numerical variable between 0 and 100 from the electroencephalogram in dogs sedated with medetomidine during dental scale removal. STUDY DESIGN: Prospective observational study. Animals Nine female Beagle dogs weighing 13.3 +/- 1.3 kg. METHODS: Cerebral state index (CSI) and burst suppression ratio (BSR) were recorded from sub-dermal needle electrodes in dogs sedated after subcutaneous injection of 114 +/- 11 microg kg(-1) medetomidine. Ten minutes after injection CSI monitoring began, and after 5 minutes, dental scale removal with an ultrasonic probe was started. After approximately 30 minutes, the effects of medetomidine were antagonized with atipamezole. RESULTS: The CSI had a median value of 43 (range 40-56) in undisturbed sedated dogs. During dental scale removal, CSI increased to a median value of 99 (range 92-100). The BSR in undisturbed sedated dogs ranged from 2 to 15, but fell to zero during dental scale removal. CONCLUSIONS: Stimulation during dental scale removal might be expected to reduce the level of sedation and hypnosis in dogs to which medetomidine had been administered. The concurrent increase in CSI and decrease in BSR suggested that a higher CSI was associated with arousal from sedation and a reduction in the depth of hypnosis. More studies are needed to validate CSI in order to better understand the functioning of this monitor. CLINICAL RELEVANCE: The CSM shows promise for monitoring the degree of sedation and hypnosis during anaesthesia, and after adequate validation, could contribute to the refinement of anaesthetic techniques in animals.

Radiotoxicity of the alpha-emitting bone-seeker 223Ra injected intravenously into mice: histology, clinical chemistry and hematology.

BACKGROUND: The alpha-emitter 223Ra, which localizes in osteoblastic active zones, including on skeletal surfaces and in osteoblastic metastases, has recently been introduced as a potential therapeutic agent against skeletal metastases. Here, the adverse effects of high dosages in animals were investigated. MATERIALS AND METHODS: Balb/c mice received intravenously (i.v.) either 1250, 2500, or 3750 kBq/kg of dissolved 223RaCl2 and were followed in the initial toxicity phase. At the 4-week end-point, the animals were sacrificed and blood samples were collected to study the effects on clinical chemistry and hematological parameters. Selected organs were weighed and tissue samples examined by microscopy. RESULTS: Treatment with 223Ra caused a dose-related minimal to moderate depletion of osteocytes and osteoblasts in the bones. Furthermore, a dose-related minimal to marked depletion of the hematopoietic cells in the bone marrow, and a minimal to slight extramedullary hematopoiesis in the spleen and in the mandibular and mesenteric lymph nodes were observed. The LD50 for acute toxicity, defined as death within 4 weeks of receiving the substance, was not reached. CONCLUSION: This study demonstrated that high doses of the bone-seeker 223Ra did not completely inactivate the blood-producing cells. The relatively high tolerance to skeletal alpha doses was probably caused by the surviving pockets of red bone marrow cells beyond the range of alpha particles from the bone surfaces, and the recruitment of peripheral stems cells.