The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions.

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called "14-3-3 site-finder". We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.

A two-step fluorinase enzyme mediated (18)F labelling of an RGD peptide for positron emission tomography.

A two-step radiolabelling protocol of a cancer relevant cRGD peptide is described where the fluorinase enzyme is used to catalyse a transhalogenation reaction to generate [(18)F]-5'-fluoro-5'-deoxy-2-ethynyladenosine, [(18)F]FDEA, followed by a 'click' reaction to an azide tethered cRGD peptide. This protocol offers efficient radiolabelling of a biologically relevant peptide construct in water at pH 7.8, 37 °C in 2 hours, which was metabolically stable in rats and retained high affinity for αVß3 integrin.

Effect of chronic antipsychotic treatment on striatal phosphodiesterase 10A levels: a [¹¹C]MP-10 PET rodent imaging study with ex vivo confirmation.

A number of phosphodiesterase 10A (PDE10) inhibitors are about to undergo clinical evaluation for their efficacy in treating schizophrenia. As phosphodiesterases are in the same signalling pathway as dopamine D2 receptors, it is possible that prior antipsychotic treatment could influence these enzyme systems in patients. Chronic, in contrast to acute, antipsychotic treatment has been reported to increase brain PDE10A levels in rodents. The aim of this study was to confirm these findings in a manner that can be translated to human imaging studies to understand its consequences. Positron emission tomography (PET) scanning was used to evaluate PDE10A enzyme availability, after chronic haloperidol administration, using a specific PDE10A ligand ([(11)C]MP-10). The binding of [(11)C]MP-10 in the striatum and the cerebellum was measured in rodents and a simplified reference tissue model (SRTM) with cerebellum as the reference region was used to determine the binding potential (BPND). In rats treated chronically with haloperidol (2 mg kg(-1) per day), there was no significant difference in PDE10A levels compared with the vehicle-treated group (BPND±s.d.: 3.57 ± 0.64 versus 2.86 ± 0.71). Following PET scans, ex vivo analysis of striatal brain tissue for PDE10A mRNA (Pde10a) and PDE10A enzyme activity showed no significant difference. Similarly, the PDE10A protein content determined by western blot analysis was similar between the two groups, contrary to an earlier finding. The results of the study indicate that prior exposure to antipsychotic medication in rodents does not alter PDE10A levels.

Unexpectedly high affinity of a novel histamine H(3) receptor antagonist, GSK239512, in vivo in human brain, determined using PET.

BACKGROUND AND PURPOSE: This study aimed to investigate the relationship between the plasma concentration (PK) of the novel histamine H3 receptor antagonist, GSK239512, and the brain occupancy of H(3) receptors (RO) in healthy human volunteers. EXPERIMENTAL APPROACH: PET scans were obtained after i.v. administration of the H(3) -specific radioligand [(11) C]GSK189254. Each subject was scanned before and after single oral doses of GSK239512, at 4 and 24 h after dose. PET data were analysed by compartmental analysis, and regional RO estimates were obtained by graphical analysis of changes in the total volumes of distribution of the radioligand, followed by a correction for occupancy by the high affinity radioligand. The PK/RO relationship was analysed by a population-modelling approach, using the average PK of GSK239512 during each scan. KEY RESULTS: Following administration of GSK239512, there was a reduction in the brain uptake of [(11) C]GSK189254 in all regions, including cerebellum. RO at 4 h was higher than at 24 h, and the PK/RO model estimated a PK associated with 50% of RO of 0.0068 ng·mL(-1) . This corresponds to a free concentration of 4.50 × 10(-12 ) M (pK = 11.3). CONCLUSIONS AND IMPLICATIONS: The affinity of GSK239512 for brain H3 receptors in humans in vivo is much higher than that expected from studies in vitro, and higher than that observed in PET studies in pigs. The study illustrates the utility of carrying out PET studies in humans early in drug development, providing accurate quantification of GSK239512 RO in vivo as a function of time and dose.

Does Early Decompressive Craniectomy Improve Outcome? Experience from an Active UK Recruiter Centre.

Introduction. The results of the recent DECRA study suggest that although craniectomy decreases ICP and ICU length of stay, it is also associated with worst outcomes. Our experience, illustrated by these two striking cases, supports that early decompressive craniectomy may significantly improve the outcome in selected patients. Case Reports. The first patient, a 20-year-old man who suffered severe brain contusion and subarachnoid haemorrhage after a fall downstairs, with refractory ICP of 35 mmHg, despite maximal medical therapy, eventually underwent decompressive craniectomy. After 18 days in intensive care, he was discharged for rehabilitation. The second patient, a 23-year-old man was found at the scene of a road accident with a GCS of 3 and fixed, dilated pupils who underwent extensive unilateral decompressive craniectomy for refractory intracranial hypertension. After three weeks of cooling, paralysis, and neuroprotection, he eventually left ICU for rehabilitation. Outcomes. Four months after leaving ICU, the first patient abseiled 40 m down the main building of St. Mary's Hospital to raise money for the Trauma Unit. He has returned to part-time work. The second patient, was decannulated less than a month later and made a full cognitive recovery. A year later, with a titanium skull prosthesis, he is back to part-time work and to playing football. Conclusions. Despite the conclusions of the DECRA study, our experience of the use of early decompressive craniectomy has been associated with outstanding outcomes. We are currently actively recruiting patients into the RESCUEicp trial and have high hopes that it will clarify the role of the decompressive craniectomy in traumatic brain injury and whether it effectively improves outcomes.

On the photochemistry of IONO2: absorption cross section (240-370 nm) and photolysis product yields at 248 nm.

The absolute absorption cross section of IONO(2) was measured by the pulsed photolysis at 193 nm of a NO(2)/CF(3)I mixture, followed by time-resolved Fourier transform spectroscopy in the near-UV. The resulting cross section at a temperature of 296 K over the wavelength range from 240 to 370 nm is given by log(10)(sigma(IONO(2))/cm(2) molecule(-1)) = 170.4 - 3.773 lambda + 2.965 x 10(-2)lambda(2)- 1.139 x 10(-4)lambda(3) + 2.144 x 10(-7)lambda(4)- 1.587 x 10(-10)lambda(5), where lambda is in nm; the cross section, with 2sigma uncertainty, ranges from (6.5 +/- 1.9) x 10(-18) cm(2) at 240 nm to (5 +/- 3) x 10(-19) cm(2) at 350 nm, and is significantly lower than a previous measurement [J. C. Mössinger, D. M. Rowley and R. A. Cox, Atmos. Chem. Phys., 2002, 2, 227]. The photolysis quantum yields for IO and NO(3) production at 248 nm were measured using laser induced fluorescence of IO at 445 nm, and cavity ring-down spectroscopy of NO(3) at 662 nm, yielding phi(IO)

Two-photon microscopy: visualization of kidney dynamics.

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.

An experimental and theoretical study of the reactions OIO+NO and OIO+OH.

The kinetics of the reaction OIO+NO were studied by pulsed laser photolysis/time-resolved cavity ring-down spectroscopy, yielding k(235-320 K)=7.6(+4.0)(-3.1) x 10(-13) exp[(607+/-128)/T] cm3 molecule-1 s-1. Quantum calculations on the OIO+NO potential-energy surface show that the reactants form a weakly bound OIONO intermediate, which then dissociates to the products IO+NO2. Rice-Ramsberger-Kassel-Markus (RRKM) calculations on this surface are in good accord with the experimental result. The most stable potential product, IONO2, cannot form because of the significant rearrangement of OIONO that would be required. The reaction OIO+OH was then investigated by quantum calculations of the relevant stationary points on its potential-energy surface. The very stable HOIO2 molecule can form by direct recombination, but the bimolecular reaction channels to HO2+IO and HOI+O2 are closed because of significant energy barriers. RRKM calculations of the HOIO2 recombination rate coefficient yield krec,0=1.5x10(-27) (T/300 K)(-3.93) cm6 molecule-2 s-1, krec,infinity=5.5x10(-10) exp(46/T) cm3 molecule-1 s-1, and Fc=0.30. The rate coefficients of both reactions are fast enough around 290 K and 1 atm pressure for these reactions to play a potentially important role in the gas phase and aerosol chemistry in the marine boundary layer of the atmosphere.

Vision and touch in ageing: crossmodal selective attention and visuotactile spatial interactions.

We investigated whether ageing affects crossmodal selective attention (the ability to focus on a relevant sensory modality and ignore an irrelevant modality) and the spatial constraints on such selective processing. Three groups of 24 participants were tested: Young (19-25 years), Young-Old (65-72 years) and Old-Old (76-92 years). The participants had to judge the elevation of vibrotactile targets (upper/index finger and lower/thumb), presented randomly to either hand while ignoring concurrent visual distractors. In a second task, the role of the target and distractor modalities was reversed. Crossmodal selective attention was assessed by comparing performance in the presence versus absence of distractors. Spatial constraints on selective attention were also investigated by comparing the effect of distractors presented on the same versus opposite side as the target. When attending to touch, the addition of visual distractors had a significantly larger effect on error rates in both of the older groups as compared to the Young group. This indicates that ageing has a detrimental effect on crossmodal selective attention. In all three age groups, performance was impaired when the target and distractor were presented at incongruent as compared to congruent elevations in both tasks. This congruency effect was modulated by the relative spatial location of the target and distractor in certain conditions for the Young and the Young-Old group. That is, participants in the two younger age groups found it harder to attend selectively to targets in one modality, when distractor stimuli came from the same side rather than from the opposite side. However, no significant spatial modulation was found in the Old-Old group. This suggests that ageing may also compromise spatial aspects of crossmodal selective attention.

Multicentre randomized-controlled clinical trial of Ipocol, a new enteric-coated form of mesalazine, in comparison with Asacol in the treatment of ulcerative colitis.

BACKGROUND: 5-Aminosalicylates remain important in the treatment of ulcerative colitis, but it is uncertain if the various preparations currently available are equivalent given the different delivery systems that exist. Generic prescription of mesalazine (mesalamine) is therefore inappropriate. Ipocol has recently become available as an alternative to Asacol-MR. AIM: To compare the two agents in a controlled trial using a non-inferiority design. METHODS: Eighty-eight ulcerative colitis patients with a mild to moderate clinical relapse were randomized to one of the two drugs at a daily dose of 2.4 g for 8 weeks. Safety was the key concern; the primary measured end-point was efficacy as judged from a colitis activity index. RESULTS: There were no unexpected adverse events of clinical consequence. The colitis score improved similarly in both patient groups (by 2.3 with Ipocol and by 1.5 with Asacol: not significant), and a similar proportion was in clinical remission at the end of the study (26.1% for Ipocol and 28.6% for Asacol: not significant). Systemic steroids were needed in 11.9% of the Asacol-treated patients compared with 6.5% with Ipocol (not significant). CONCLUSION: It appears appropriate to conclude that, while not identical to Asacol-MR, Ipocol offers a safe and similarly effective alternative.

Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells.

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.

Light-emitting diode (LED) polymerisation of dental composites: flexural properties and polymerisation potential.

The clinical performance of light polymerised dental composites is greatly influenced by the quality of the light-curing unit (LCU) used. Commonly used halogen LCUs have some specific drawbacks such as decreasing of the light output with time. This may result in low degree of monomer conversion of the composites with negative clinical implications. Previous studies have shown that blue-light-emitting diode (LED) LCUs have the potential to polymerise dental composites without having the drawbacks of halogen LCUs. Despite the relatively low irradiance of current LED LCUs, their efficiency is close to that of conventional halogen LCUs with more than twice the irradiance. This phenomenon has not been explained fully yet. Hence, more tests of the LED LCU's effectiveness and of the mechanical properties of oral biomaterials processed with LED LCUs need to be carried out. This study investigates the flexural properties of three different composites with three different shades, which were polymerised with either a commercial halogen LCU or an LED LCU, respectively. In most cases no significant differences in flexural strength and modulus between composites polymerised with a halogen LCU or an LED LCU, respectively, were found. A simple model for the curing effectiveness based on the convolution absorption spectrum of the camphorquinone photoinitiator present in composites and the emission spectra of the LCUs is presented.

Depth of cure and compressive strength of dental composites cured with blue light emitting diodes (LEDs).

OBJECTIVE: The primary objective of this pilot study was to test the hypotheses that (i) depth of cure and (ii) compressive strength of dental composites cured with either a light emitting diode (LED) based light curing unit (LCU) or a conventional halogen LCU do not differ significantly. The second objective of this study was to characterise irradiance and the emitted light spectra for both LCUs to allow comparisons between the units. METHODS: Dental composite (Spectrum TPH, shades A2 and A4) was cured for 40 s with either a commercial halogen LCU or a LED LCU, respectively. The LED LCU uses 27 blue LEDs as the light source. The composites' depth of cure was measured for 10 samples of 4 mm diameter and 8 mm depth for each shade with a penetrometer. The results were compared using a Student's t-test. Compressive strengths were determined after 6 and 72 h, for six samples of 4 mm diameter and 6 mm depth for each shade after being polymerised for 40 s from each end of the mould. Groups were compared using a three way ANOVA. RESULTS: The conventional halogen LCU cured composites significantly (p < 0.05) deeper (6.40 mm A2, 5.19 mm A4) than did the LED LCU (5.33 mm A2, 4.27 mm A4). Both units cured the composite deeper than required by both ISO 4049 and the manufacturer. A three way ANOVA showed that there were no significant differences in the compressive strengths of samples produced with either the LED LCU or the halogen LCU (p = 0.460). Significant differences in compressive strength of samples stored for 6 and 72 h (p = 0.0006) and of samples of different shades (p = 0.035) were found as confirmed by the three way ANOVA. The light spectra of both units differed strongly. While the halogen LCU showed a broad distribution of wavelengths with a power peak at 497 nm, the LED LCU emitted most of the generated light at 465 nm. The LED LCU produced a total irradiance of 350 mW cm-2 whereas the halogen LCU produced a total irradiance of 755 mW cm-2. SIGNIFICANCE: The results showed that both units provided sufficient output to exceed minimum requirements in terms of composites' depth of cure according to ISO 4049 and the depth of cure and the composites' compressive strength stated by the manufacturer. Compressive strengths of dental composites cured under laboratory conditions with a LED LCU were statistically equivalent to those cured with a conventional halogen LCU. With its inherent advantages, such as a constant power output over the lifetime of the diodes, LED LCUs have great potential to achieve a clinically consistent quality of composite cure.

Pathophysiology and functional significance of apical membrane disruption during ischemia.

The characteristic structure of polarized proximal tubule cells is drastically altered by the onset of ischemic acute renal failure. Distinctive apical brush border microvilli disruption occurs rapidly and in a duration-dependent fashion. Microvillar membranes internalize into the cytosol of the cell or are shed into the lumen as blebs. The microvillar actin core disassembles concurrent with or preceding these membrane changes. Actin and its associated binding proteins no longer interact to form these highly regulated apical membrane structures necessary for microvilli. The resultant epithelial cells have a reduced apical membrane surface that is not polarized either structurally, biochemically or physiologically. Furthermore, the changes in the apical microvilli result in tubular obstruction, reduced Na+ absorption, and partly explain the reduction in glomerular filtration rate. Recent evidence suggests these actin surface membrane alterations induced by ischemia are secondary to activation and relocation of the actin-associated protein, actin depolymerizing factor/cofilin, to the apical membrane domain. Activated (dephosphorylated) actin depolymerizing factor/cofilin proteins bind filamentous actin, increasing subunit treadmilling rates and filament severing. Once activated, the diffuse cytoplasmic distribution of the actin depolymerizing factor/cofilin protein relocalizes to the luminal membrane blebs. During recovery the actin depolymerizing factor/cofilin proteins are again phosphorylated and reassume their normal diffuse cytoplasmic localization. This evidence strongly supports the hypothesis that actin depolymerizing factor/cofilin proteins play a significant role in ischemia-induced injury in the proximal tubule cells.

Dental composite depth of cure with halogen and blue light emitting diode technology.

OBJECTIVES: To test the hypothesis that a blue light emitting diode (LED) light curing unit (LCU) can produce an equal dental composite depth of cure to a halogen LCU adjusted to give an irradiance of 300 mWcm-2 and to characterise the LCU's light outputs. MATERIALS AND METHODS: Depth of cure for three popular composites was determined using a penetrometer. The Student's t test was used to analyse the depth of cure results. A power meter and a spectrometer measured the light output. RESULTS: The spectral distribution of the LCUs differed strongly. The irradiance for the LED and halogen LCUs were 290 mWcm-2 and 455 mWcm-2, when calculated from the scientific power meter measurements. The LED LCU cured all three dental composites to a significantly greater (P < 0.05) depth than the halogen LCU. CONCLUSIONS: An LED LCU with an irradiance 64% of a halogen LCU achieved a significantly greater depth of cure. The LCU's spectral distribution of emitted light should be considered in addition to irradiance as a performance indicator. LED LCUs may have a potential for use in dental practice because their performance does not significantly reduce with time as do conventional halogen LCUs.

Density-dependent effects on Anguillicola crassus (Nematoda) within its European eel definitive host.

Density-dependent effects on adult and larval stages of the introduced nematode parasite Anguillicola crassus in the European eel definitive host were examined in the laboratory in naturally infected fish, and in field samples. The effect of adult nematode subpopulations on larval development over time and the effect of increasing adult intensity on the gravid female subpopulations were investigated. At high adult subpopulations the movement of larvae from the swimbladder wall into the swimbladder lumen appears to be inhibited, and A. crassus larvae to be arrested in development in a density-dependent manner. The number of gravid females per host reaches a constant level, and so the proportion of gravid female nematodes per adult subpopulation decreases relative to further increasing total adult nematode numbers. Both these mechanisms have the potential to regulate infrapopulations of A. crassus within the eel definitive host and thus the parasite suprapopulation.

Modulatory effects of L-DOPA on D2 dopamine receptors in rat striatum, measured using in vivo microdialysis and PET.

Putative modulatory effects of L-3,4-dihydroxyphenylalanine (L-DOPA) on D2 dopamine receptor function in the striatum of anaesthetised rats were investigated using both in vivo microdialysis and positron emission tomography (PET) with carbon-11 labelled raclopride as a selective D2 receptor ligand. A single dose of L-DOPA (20 or 100mg/kg i.p.) resulted in an increase in [11C]raclopride binding potential which was also observed in the presence of the central aromatic decarboxylase inhibitor NSD 1015, confirming that the effect was independent of dopamine. This L-DOPA evoked D2 receptor sensitisation was abolished by a prior, long-term administration of L-DOPA in drinking water (5 weeks, 170mg/kg/day). In the course of acute L-DOPA treatment (20mg/kg), extracellular GABA levels were reduced by approximately 20% in the globus pallidus. It is likely that L-DOPA sensitising effect on striatal D2 receptors, as confirmed by PET, may implicate striato-pallidal neurones, hence a reduced GABA-ergic output in the projection area. Since the L-DOPA evoked striatal D2 receptor supersensitivity habituates during long-term treatment, the effects reported here may contribute to the fluctuations observed during chronic L-DOPA therapy in Parkinson's disease.

In vivo saturation kinetics of two dopamine transporter probes measured using a small animal positron emission tomography scanner.

When estimated in vitro, the parameters which describe the binding of radiolabelled analogues of cocaine to sites on the dopamine transporter are very much influenced by the methodology used. In the present study, a small animal positron emission tomography (PET) scanner was used to estimate in vivo saturation kinetics for two carbon-11 labelled compounds presently used to monitor dopamine terminal function. The binding of [11C]CFT (WIN 35,428) in rat striatum was adequately described by a single-site model, giving an apparent dissociation constant corresponding to an intravenous dose of 242 nmol/kg. In contrast, the binding of [11C]RTI-121 was better described by a two-site model with the 'high-affinity' site or state (dissociation constant = 1 nmol/kg) being significantly occupied at doses routinely used in PET scanning. Such findings cannot readily be predicted from in vitro work, but could aid in both the choice of ligand and the model used in quantification of scan data. While multi-dose in vivo PET studies are difficult in man, rat PET can easily be employed either pre-clinically for putative radioligands, or experimentally, to study drug interactions and receptor occupancy related to functional efficacy.

The effects of donor stage on the survival and function of embryonic striatal grafts in the adult rat brain. II. Correlation between positron emission tomography and reaching behaviour.

Grafts of embryonic striatal primordia are able to elicit behavioural recovery in rats which have received an excitotoxic lesion to the striatum, and it is believed that the P zones or striatal-like tissue within the transplants play a crucial role in these functional effects. We performed this study to compare the effects of different donor stage of embryonic tissue on both the morphology (see accompanying paper) and function of striatal transplants. Both the medial and lateral ganglionic eminence was dissected from rat embryos of either 10 mm, 15 mm, 19 mm, or 23 mm crown-rump length, and implanted as a cell suspension into adult rats which had received an ibotenic acid lesion 10 days prior to transplantation. After four months the animals were tested on the "staircase task" of skilled forelimb use. At 10-14 months rats from the groups which had received grafts from 10 mm or 15 mm donor embryos were taken for positron emission tomography scanning in a small diameter positron emission tomography scanner, using ligands to the dopamine D1 and D2 receptors, [11C]SCH 23390 and [11C]raclopride, respectively. A lesion-alone group was also scanned with the same ligands for comparison. Animals which had received transplants from the 10 mm donors showed a significant recovery with their contralateral paw on the "staircase test". No other groups showed recovery on this task. Similarly, the animals with grafts from the youngest donors showed a significant increase in D1 and D2 receptor binding when compared to the lesion-alone group. No increase in signal was observed with either ligand in the group which had received grafts from 15 mm donors. Success in paw reaching showed a strong correlation to both the positron emission tomography signal obtained and the P zone volume of the grafts. These results suggest that striatal grafts from younger donors (10 mm CRL) give greater behavioural recovery than grafts prepared from older embryos. This recovery is due to both the increased proportion of striatal-like tissue within the grafts and an increase in functional D1 and D2 dopamine receptors measured by positron emission tomography, i.e. a more extensive integration of the graft with the host brain.

Actin Purified from Maize Pollen Functions in Living Plant Cells.

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 [alpha]-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 [alpha]-actin and had a critical concentration for assembly of 0.6 [mu]M. 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.