No abstract

The histamine H 3 receptor is implicated in the pathophysiology of several central nervous system disorders. N-methyl-6-(3-cyclobutyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yloxy)-nicotamide (GSK189254) is a highly potent, selective, and brain-penetrant H 3 receptor antagonist. Previous studies in the pig using PET have shown that 11 C-GSK189254 uptake in H 3 -rich regions of the brain can be blocked by the selective H 3 antagonist ciproxifan. The purpose of the present study was to evaluate 11 C-GSK189254 as a PET radioligand for human studies and to determine the dose-receptor occupancy relationship of GSK189254 in the human brain. Methods: Dynamic PET scans were obtained in healthy subjects over 90 min after intravenous administration of approximately 370 MBq of 11 C-GSK189254. Blood samples were taken throughout the scans to derive the arterial plasma parent input function. Each subject was scanned twice, either with tracer alone (test-retest) or before and after a single oral dose of GSK189254 (10-100 mg). Data were analyzed by compartmental analysis, and regional receptoroccupancy estimates were obtained by graphical analysis of changes in the total volumes of distribution (V T ) of the radioligand. Results: 11 C-GSK189254 readily entered the brain; its regional brain distribution reflected the known distribution of H 3 receptors, with high binding in the caudate and putamen, intermediate binding in cortical regions, and low binding in the cerebellum. GSK189254 displayed a high receptor affinity, and a marked reduction in V T was apparent at all the doses tested. The oral dose equaling 50% occupancy of the available receptor sites (ED 50 ) was estimated as 4.33 mg. Additional data on plasma pharmacokinetics after oral dosing and the plasma free fraction gave a corresponding estimate of the free concentration of GSK189254 required to occupy 50% of the available receptor sites (EC 50 ) (0.011 nM). The test-retest data showed reductions in regional V T on the second scan in all subjects. A nonlinear compartmental analysis of this effect demonstrated that this reduction was consistent with carryover of a tracer mass dose effect with an estimated in vivo apparent dissociation constant of 0.010 nM, close to the independent estimate of the plasma EC 50 . Conclusion: 11 C-GSK189254 can be used to quantify H 3 receptor availability in humans in vivo using PET but requires high specific activity; the possibility of tracer mass dose effects should be carefully analyzed.

The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.

BackgroundPodocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration.Methods and FindingsWe evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus.ConclusionsBased on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.

Microglial activation has been linked with deficits in neuronal function and synaptic plasticity in Alzheimer's disease (AD). The mitochondrial translocator protein (TSPO) is known to be upregulated in reactive microglia. Accurate visualization and quantification of microglial density by PET imaging using the TSPO tracer [(11)C]-R-PK11195 has been challenging due to the limitations of the ligand. In this study, it was aimed to evaluate the new TSPO tracer [(11)C]PBR28 as a marker for microglial activation in the 5XFAD transgenic mouse model of AD. Dynamic PET scans were acquired following intravenous administration of [(11)C]PBR28 in 6-month-old 5XFAD mice and in wild-type controls. Autoradiography with [(3)H]PBR28 was carried out in the same brains to further confirm the distribution of the radioligand. In addition, immunohistochemistry was performed on adjacent brain sections of the same mice to evaluate the co-localization of TSPO with microglia. PET imaging revealed that brain uptake of [(11)C]PBR28 in 5XFAD mice was increased compared with control mice. Moreover, binding of [(3)H]PBR28, measured by autoradiography, was enriched in cortical and hippocampal brain regions, coinciding with the positive staining of the microglial marker Iba-1 and amyloid deposits in the same areas. Furthermore, double-staining using antibodies against TSPO demonstrated co-localization of TSPO with microglia and not with astrocytes in 5XFAD mice and human post-mortem AD brains. The data provided support of the suitability of [(11)C]PBR28 as a tool for in vivo monitoring of microglial activation and assessment of treatment response in future studies using animal models of AD.

A strategy for last-step (18)F fluorination of bioconjugated peptides is reported that exploits an "Achilles heel" in the substrate specificity of the fluorinase enzyme. An acetylene functionality at the C-2 position of the adenosine substrate projects from the active site into the solvent. The fluorinase catalyzes a transhalogenation of 5'-chlorodeoxy-2-ethynyladenosine (ClDEA) to 5'-fluorodeoxy-2-ethynyladenosine (FDEA). Extending a polyethylene glycol linker from the terminus of the acetylene allows the presentation of bioconjugation cargo to the enzyme for (18)F labelling. The method uses an aqueous solution (H2(18)O) of [(18)F]fluoride generated by the cyclotron and has the capacity to isotopically label peptides of choice for positron emission tomography (PET).

A positron emission tomograph (PET) was used to image D2 dopamine receptor function in rat striata and to obtain regional time-radioactivity curves from individual rat brains following i.v. injection of carbon-11-labelled raclopride. Despite the limited resolution of the camera, together with associated spillover and partial volume effects, the kinetic data obtained from striata were such that specific binding of the radioligand could be quantified unilaterally, using a reference tissue compartmental model, with cerebellum data as an indirect input function. With the exception that the rat is anaesthetised, the experimental system is analogous to the acquisition and collection of clinical PET data and, by using animal models of disease, can be used to aid the interpretation of clinical studies. Using 6-hydroxydopamine (6-OHDA) lesioning of the substantia nigra pars compacta to produce a rat hemiparkinsonian model, the present results confirm that deafferentation causes a supersensitivity of post-synaptic D2 dopamine receptors. Saturation studies indicated that the measured 23% increase in [11C]raclopride binding potential reflected a change in receptor affinity. Modulation of extracellular dopamine concentration, monitored by in vivo microdialysis, demonstrated that the increased binding was unlikely to be due to a reduction in receptor occupancy by endogenous dopamine. Acute administration of L-3,4-dihydroxyphenylalanine (L-dopa) also caused an increase in [11C]raclopride binding potential, confirming the suggestion that L-dopa plays a more complex role than that of dopamine precursor in the nigrostriatal pathway.

A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal a-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate a-actin and had a critical concentration for assembly of 0.6 pM. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.