Description of an XRF system for multielemental analysis

An X-ray fluorescence (XRF) system that uses radioisotopes in an orthogonal configuration between the source, sample, and detector is described. The advantage of such a system is that for large (bulk) samples or in vivo measurements, the background caused by Compton scattering in the sample is minimized. High reproducibility for nonuniform samples is obtained by reducing the sample size and thus the effects of nonuniformity in the spatial response of such a system. Germane to any accurate analytical method is the use of proper mathematical algorithms for data evaluation. The problem is acute, in particular, when photopeaks with low counting statistics are to be analyzed. In the case of a single photopeak on flat background, optimal energy window size, which maximizes the signal-to-noise ratio, for trapezoidal intergration is described. The sensitivity and minimum detection limit at different energies together with background considerations are discussed.     

N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 2. Fast-reaction studies show that a fluorescent substrate analogue binds independently to two different sites.

The mechanism of binding of reduced 1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate (rCdRP) to two different binding sites on the bifunctional enzyme is determined by kinetic studies, using temperature-jump and stopped-flow equipment with fluorescence detection. Two rapid binding processes and a comparatively slow isomerization process are observed over a wide range of enzyme and rCdRP concentrations. Kinetic measurements with low concentrations of rCdRP show that the isomerization is coupled only to the more rapid of the two binding reactions that involves the active site of indoleglycerol-phosphate synthase. The slower of the two binding reactions represents rCdRP binding in one step to the active site of (phosphoribosyl)anthranilate isomerase. The simplest mechanism explaining quantitatively the dependence of the relaxation times on concentration consists of rCdRP binding to two sites on the enzyme that are intrinsically different and independent, even to the extent that a ligand-induced isomerization of one site is not transmitted to the other site. Simulation studies show that the concentration dependences of the amplitudes of the three relaxation processes are also consistent with the mechanism. The results are discussed in terms of two autonomous domains of folding of the polypeptide chain.     

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro–computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7^th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7^th and 10^th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.     

Human Nek6 is a monomeric mostly globular kinase with an unfolded short N-terminal domain

Background  

The NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they represent an evolutionarily conserved family of 11 serine/threonine kinases, with 40-45% amino acid sequence identity to the Aspergillus nidulans mitotic regulator NIMA within their catalytic domains. Neks have cell cycle-related functions and were recently described as related to pathologies, particularly cancer, consisting in potential chemotherapeutic targets. Human Nek6, -7 and -9 are involved in the control of mitotic spindle formation, acting together in a mitotic kinase cascade, but their mechanism of regulation remain elusive.