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Traumatic brain injury (TBI) represents a major cause of death and disability in early adulthood. Concomitant extracranial injury such as long bone fracture was reported to exacerbate TBI pathology. However, early reciprocal effects and mechanisms have been barely investigated. To address this issue, C57BL/6N mice were subjected to either the controlled cortical impact (CCI) model of TBI, fracture of the left femur (FF), combined injury (CCI+FF), or sham procedure. Behavioral alterations were monitored until 5 days post injury (dpi), followed by (immuno-)histology, gene and protein expression analyses using quantitative PCR, western blot, and ELISA. We found that CCI+FF mice exhibited increased neurological impairments, reduced recovery, and altered anxiety-related behavior compared to single injury groups. At 5 dpi, cerebral lesion size was not affected by combined injury but exaggerated hippocampal substance loss and increased perilesional astrogliosis were observed in CCI+FF mice compared to isolated CCI. Bone gene expression of the osteogenic markers Runx2, osteocalcin, alkaline phosphatase, and bone sialoprotein was induced by fracture injury but attenuated by concomitant TBI. Plasma concentrations of the biomarkers osteopontin and progranulin were elevated in CCI+FF mice compared to other experimental groups. Taken together, using a murine model of TBI and femoral fracture, we report early reciprocal impairments of brain tissue maintenance, behavioral recovery, and bone repair gene expression. Increased circulating levels of the biomarkers osteopontin and progranulin indicate ongoing tissue inflammation and repair. Our results may have implications for future therapeutic approaches to interfere with the pathological crosstalk between TBI and concomitant bone fracture. [ABSTRACT FROM AUTHOR]

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. [ABSTRACT FROM AUTHOR]

Transhydrogenase is a proton-pumping membrane protein that is required for the cellular regeneration of NADPH. The NADPH- binding domain (domain I) of transhydrogenase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique at room temperature. The crystals. which were grown from PFG 4000 and ammonium acetate in citrate buffer, belong to the triclinic space group P1, with unit-cell parameters a = 38.8, b = 68.8, c = 76.4 Å, α = 67.5, β = 80.8, γ = 81.5. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The crystals contain one dimer of transhydrogenase domain I per asymmetric unit. [ABSTRACT FROM AUTHOR]

Hydrolysis is an often-encountered obstacle in the crystallization of proteins complexed with their substrates. As the duration of the crystallization process, from nucleation to the growth of the crystal to its final size, commonly requires several weeks, non-enzymatic hydrolysis of an "unstable" ligand occurs frequently. In cases where the crystallization conditions exhibit non neutral pH values this hydrolysis phenomenon may be even more pronounced. ChoX, the substrate binding protein of a choline ABC-importer, produced crystals with its substrate acetylcholine after one month. However, these crystals exhibited only choline, an acetylcholine hydrolysis product, in the binding site. To overcome this obstacle we devised a microseeding protocol leading to crystals of ChoX with bound acetylcholine within 24 hours. One drawback we encountered was the high twinning fraction of the crystals, possibly was due to the rapid crystal growth.