Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A methodology for the rapid and quantitative analysis of phosphorylation sites in proteins is presented. The coupling of capillary high-performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) allowed one to distinguish phosphorylation sites based on retention time and mass difference from complex peptide mixtures. The methodology was first evaluated and validated for a mixture of non-, mono-, and dityrosine-phosphorylated synthetic peptides, corresponding to the tryptic fragment 485–496 (ALGADDSYYTAR) of the human protein tyrosine kinase ZAP-70. The limits of detection for the non-, mono- and diphosphorylated peptides were about 15, 40 and 100 fmol, respectively, when using a 300 μm I.D. column. Application of the method was extended to identify phosphopeptides generated from a trypsin digest of recombinant autophosphorylated ZAP-70, in particular with respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass spectrometry data allowed one to ascertain the identity of the detected peptides, a prerequisite to analyses in more complex biological samples. As an extension to the methodology described above, we evaluated the feasibility of interfacing capillary HPLC to matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), using a micromachined piezoelectric flow-through dispenser as the interface. This enabled direct arraying of chromatographically separated components onto a target plate that was precoated with matrix for subsequent analysis by MALDI-TOF-MS without further sample handling.     

Topographical distribution of purine nucleoside phosphorylase in the human neuraxis

The activity of purine nucleoside phosphorylase was determined at various levels of the human neuraxis in 5 brains and 2 spinal cords, using the method of Lewis and Glantz. The determination is based on the decrease in optical density of guanosine at 252 nm and 40 degrees C, with conversion of this compound to guanine and ribose-1-phosphate by phosphorolysis. Our studies show a fairly uniform distribution of the enzyme in the human CNS, with an average value of 209 mumol of guanosine transformed/min/g of wet tissue. The lowest values are found in the spinal cord and cerebellar grey matter, and highest amounts in the occipital grey and white substance.     

Novel Nano-Sized MR Contrast Agent Mediates Strong Tumor Contrast Enhancement in an Oncogene-Driven Breast Cancer Model

The current study was carried out to test the potential of a new nanomaterial (Spago Pix) as a macromolecular magnetic MR contrast agent for tumor detection and to verify the presence of nanomaterial in tumor tissue. Spago Pix, synthesized by Spago Nanomedical AB, is a nanomaterial with a globular shape, an average hydrodynamic diameter of 5 nm, and a relaxivity (r₁) of approximately 30 (mM Mn)⁻¹ s⁻¹ (60 MHz). The material consists of an organophosphosilane hydrogel with strongly chelated manganese (II) ions and a covalently attached PEG surface layer. In vivo MRI of the MMTV-PyMT breast cancer model was performed on a 3 T clinical scanner. Tissues were thereafter analyzed for manganese and silicon content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The presence of nanomaterial in tumor and muscle tissue was assessed using an anti-PEG monoclonal antibody. MR imaging of tumor-bearing mice (n = 7) showed a contrast enhancement factor of 1.8 (tumor versus muscle) at 30 minutes post-administration. Contrast was retained and further increased 2–4 hours after administration. ICP-AES and immunohistochemistry confirmed selective accumulation of nanomaterial in tumor tissue. A blood pharmacokinetics analysis showed that the concentration of Spago Pix gradually decreased over the first hour, which was in good agreement with the time frame in which the accumulation in tumor occurred. In summary, we demonstrate that Spago Pix selectively enhances MR tumor contrast in a clinically relevant animal model. Based on the generally higher vascular leakiness in malignant compared to benign tissue lesions, Spago Pix has the potential to significantly improve cancer diagnosis and characterization by MRI.     

An Integrated Understanding of the Rapid Metabolic Benefits of a Carbohydrate-Restricted Diet on Hepatic Steatosis in Humans

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Five classes of visual interneurons in the optic nerve of the hermit crab

Response characteristics and trigger features are described for five operational classes of visual interneurons in the optic nerve of the hermit crab. The units were initially segregated on the basis of response sign (“on”, “off”, “on-off”) and duration (phasic, tonic). Tonic “on” and tonic “off” units were observed to be indistinguishable from the previously described sustaining and dimming fibers of other decapod crustacea. Phasic “off” units closely resemble either jittery motion or novelty detectors also previously documented. Phasic “on” units are characterized by an excitable antagonistic surround, a slow rate of habituation, and a sustained low level discharge during periods of increasing luminous flux at any point within their receptive fields. Highly sensitive to moving contours, they occasionally revealed a directional preference. The phasic “on” units are also capable of following brief stimuli at rates of up to 20/sec. Phasic “on-off” units were characterized by coextensive receptive fields for “on” and “off” responses and movement sensitivity independent of contrast.     

Purification and properties of human liver monoamine oxidase

Human liver monoamine oxidase [monoamine: O₂ oxidoreductase (deaminating), E. C. 1.4.3.4] was purified by a method which does not depend on the isolation of mitochondria, and in which vacuum dialysis, during which the enzyme separates out as a yellow precipitate, is an important step in purification. By this method a final specific activity of 550 and fold purification of 40 was attained. A single peak was obtained with the analytical ultracentrifuge, and a sedimentation constant of 6.78S noted. A single active band was observed by polyacrylamide gel electrophoresis. The enzyme exhibits optimum activity at pH 8.7, with no activity below pH 5.5 or above pH 11.8. Using benzylamine hydrobromide as the substrate, in 0.05 m phosphate buffer (pH 7.4) at 27 °C, the Michaelis constant was found to be 1.7 × 10^?3m. The enzyme, which is quite stable, is a flavo-protein, as shown by absorption and fluorescence spectra. The C-terminal group is glycine. The molecular weight, as determined by SDS polyacrylamide-gel electrophoresis, is 64,000. Repeated attempts to determine the N-terminal group were unsuccessful.