The accuracy of magnetic resonance imaging in prostate cancer staging: a single-institution experience.

BACKGROUND: Magnetic resonance imaging (MRI) has a wide reported variation in sensitivity and specificity for staging prostate cancer (PCA). AIMS: We examined the accuracy of MRI in detecting PCA, and in identifying extracapsular extension (ECE) and seminal vesicle invasion (SVI) in PCA patients at our institution. METHODS: We retrospectively reviewed pre-biopsy MRI findings and correlated the same with subsequent radical prostatectomy pathology reports in all patients undergoing radical prostatectomy between 2010 and 2012. Specifically, comparison was made between MRI and pathologic stage. Age, serum prostate-specific antigen level and Gleason score were recorded. RESULTS: MRI detected signal abnormalities in 50 out of 88 PCA patients undergoing radical prostatectomy. Of these, 12 had ECE and 7 had SVI on final histology. The sensitivity and specificity of MRI for detecting ECE were 75 and 100%, respectively. The sensitivity and specificity of MRI for detecting SVI were 16.7 and 100%, respectively. The positive predictive values for determining ECE and SVI were 100% and negative predictive values were 96.2 and 90.6%, respectively. CONCLUSIONS: MRI may be reliable for excluding ECE and SVI in PCA patients where the lesion is visible on MRI. It has a good diagnostic ability for ECE, but is less accurate for identifying SVI. This article supports the use of MRI in the preoperative evaluation of PCA.

Nucleophosmin is a novel Bax chaperone that regulates apoptotic cell death.

The proapoptotic B-cell lymphoma-2 family protein Bax is a key regulatory point in the intrinsic apoptotic pathway. However, the factors controlling the process of Bax activation and translocation to mitochondria have yet to be fully identified and characterized. We performed affinity chromatography using peptides corresponding to the mitochondrial-targeting region of Bax, which is normally sequestered within the inactive structure. The molecular chaperone nucleophosmin was identified as a novel Bax-binding protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Reciprocal co-immunoprecipitation and proximity assays confirmed the Bax-nucleophosmin protein-protein interaction and verified that nucleophosmin only bound to activated conformationally altered Bax. Confocal microscopy in a cell-based apoptosis model, demonstrated that nucleophosmin translocation from nucleolus to cytosol preceded Bax movement. Specific knockdown of nucleophosmin expression using RNAi attenuated apoptosis as measured by mitochondrial cytochrome c release and activation of the caspase cascade. In a mouse model of ischaemic stroke, subcellular fractionation studies verified that nucleophosmin translocation occurred within 3 h, at a time before Bax translocation but after Bax conformational changes have occurred. Thus, we have elucidated a novel molecular mechanism whereby Bax becomes activated and translocates to the mitochondria to orchestrate mitochondrial dysfunction and apoptotic cell death, which opens new avenues for therapeutic intervention.

Differential regulation of caspase-3 by pharmacological and developmental stimuli as demonstrated using humanised caspase-3 mice.

Caspase-3 is a potential therapeutic target for a number of degenerative diseases. However the development of specific caspase-3 inhibitors has been hampered by inter-species differences and the high degree of homology shared by different caspases. To circumvent these issues, we have produced and characterised a humanised caspase-3 mouse line (possessing one copy of the human gene with both copies of the murine gene disrupted) by crossing human caspase-3 transgenic mice with nullizygous caspase-3 knock-out mice. Humanised mice appeared normal and survived to adulthood. Analysis of the human gene revealed that human pro-caspase-3 was expressed in the same tissues as its murine counterpart. However humanised mice retained the hypercellularity of frontal cortex seen in their knock-out parental line and there was no biochemical evidence of human protein processing during naturally occurring neuronal death taking place during brain development. In contrast, the human protein was cleaved by the mouse machinery following anti-Fas treatment of adult mice. These data suggest that there is a fundamental difference between the activation pathways leading to caspase-3 cleavage during naturally occurring cell death in development/embryogenesis and following an apoptotic stimulus in the adult.

Mice overexpressing human caspase 3 appear phenotypically normal but exhibit increased apoptosis and larger lesion volumes in response to transient focal cerebral ischaemia.

Caspase 3 activation has been implicated in cell death following a number of neurodegenerative insults. To determine whether caspase genes can affect the susceptibility of cells to neurodegeneration, a transgenic mouse line was created, expressing human caspase 3 under control of its own promoter. The human gene was regulated by the murine homeostatic machinery and human procaspase 3 was expressed in the same tissues as mouse caspase 3. These novel transgenic mice appeared phenotypically and developmentally normal and survived in excess of 2 years. Behavioural assessment using the 5-choice serial reaction time task found no differences from wild-type littermates. Caspase activity was found to be tightly regulated under physiological conditions, however, significantly larger lesions were obtained when transgenic mice were subjected to focal cerebral ischaemia/reperfusion injury compared to wild-type littermates. These data demonstrate that mice overexpressing human caspase 3 are essentially normal, however, they have increased susceptibility to degenerative insults.

[3H]dofetilide binding to HERG transfected membranes: a potential high throughput preclinical screen.

The pharmacological characteristics of [3H]dofetilide binding were examined in membranes prepared from human embryonic kidney (HEK293) cells stably expressing human ether-á-go-go related gene (HERG) K+ channels. The classIII antiarrhythmic compounds dofetilide, clofilium, 4'-[[1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidyl]carbonyl]methanesulfonanilide (E-4031), N-methyl-N-[2-[methyl-(1-methyl-1H-benzimidazol-2-yl)amino]ethyl]-4-[(methylsulfonyl)amino]benzene-sulfonamide (WAY-123,398) and d-sotalol all inhibited [3H]dofetilide binding. In addition, the structurally unrelated compounds pimozide, terfenadine and haloperidol, all of which prolong the QT interval in man, also inhibited binding. These data indicate that a [3H]dofetilide binding assay using HERG membranes may help identify compounds that prolong the QT interval.

Phenotyping complex behaviours: assessment of circadian control and 5-choice serial reaction learning in the mouse.

Currently, the behavioural phenotyping of mutant strains is restricted by the paucity of tests for the cognitive capabilities of mice. Most of the paradigms at present available such as the water maze or passive avoidance are dependent upon an aversive component for conditioning and as a consequence the data can often be confounded by the non-specific effects of stress. The development of mutant or transgenic mouse models of human diseases that effect cognitive function will require this confound to be overcome. Here we highlight the value of using two apparently different paradigms, one exploring circadian control and the other visuospatial attention. The first provides an example of an elicited behaviour while the second requires learning; neither is aversive in nature. Interestingly, the data from each strengthens the interpretation of both.

Characterisation of [(125)I]-apamin binding sites in rat brain membranes with HE293 cells transfected with SK channel subtypes.

The pharmacology of [(125)I]-apamin binding sites was examined in rat cortical and hippocampal tissue and compared with membranes prepared from human embryonic kidney (HEK293) cells transfected with SK channel subtypes hSK1, rSK2 and rSK3. The K(D) of [(125)I]-apamin in rat cortex and hippocampus was similar to the apamin-sensitive subtypes, rSK2 and rSK3 (K(D) (pM): 6.4, 7.08, 6.56 and 8.94, respectively). In addition, [(125)I]-apamin had a K(D)=270.4pM for the putatively 'apamin-insensitive' hSK1. Apamin had about a three-fold higher affinity than [(125)I]-apamin in brain tissue and in the cells expressing the different SK channel subtypes. Pancuronium, bicuculline and d-tubocurarine displayed micromolar affinity for all five-membrane preparations, whereas dequalinium and gallamine appear to show some subtype selectivity. Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) had millimolar affinity and linopirdine had no effect. In conclusion, the pharmacology of [(125)I]-apamin binding in the cortex and hippocampus was similar to that in the apamin-sensitive clones, rSK2 and rSK3. In addition, we demonstrated low affinity [(125)I]-apamin binding for hSK1 and identified compounds that show subtype selectivity. These data cast further doubt on the identification of SK1 as encoding for the K(+) channel responsible for the apamin-insensitive sAHP.

Modulation of the hyperpolarisation-activated current, Ih, in rat facial motoneurones in vitro by ZD-7288.

ZD-7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride] and Cs(+) have been used to distinguish the currents contributing to inward rectification in neonatal rat facial motoneurones (FMs). ZD-7288 (0.1-10 microM) inhibited a current that reversed at -43.7+/-3.7 mV in artificial cerebrospinal fluid (ACSF) containing 3 mM K(+) (n=9), and displayed the time and voltage dependence of the hyperpolarisation-activated current, I(h). Depolarisation-activated transient (I(K(A))) and sustained outward currents were unaffected by ZD-7288. The IC(50) for block of I(h) by ZD-7288 was around 0.2 microM. Onset of inhibition was slow and no recovery was seen after washing in ZD-7288-free ACSF for up to 4 h. In the presence of ZD-7288, Ba(2+) and Rb(+) blocked an inwardly rectifying potassium (K(+)) current, confirming both the presence of I(K(IR)) and its insensitivity to ZD-7288. Cs(+) rapidly and reversibly blocked both I(h) and I(K(IR)). Inhibition of I(h) by ZD-7288 showed no use dependence, internally applied ZD-7288 also blocked I(h), and tail current analysis indicated inhibition to be voltage-independent. In the presence of internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and after previous exposure to ZD-7288, 5-hydroxytryptamine (5-HT), but not noradrenaline, promoted a recovery of I(h) that was not observed if ZD-7288 was present throughout the recording period. This interaction between ZD-7288 and irreversible 5-HT-receptor activation may be related to the mechanism underlying ZD-7288-mediated block of these channels.

Effect of FK960, a putative cognitive enhancer, on synaptic transmission in CA1 neurons of rat hippocampus.

The action of FK960 [N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate], a novel cognitive enhancer, on excitatory synaptic transmission in the hippocampus was investigated. Excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were recorded intracellularly from CA1 neurons in rat hippocampus using the "blind patch" variant of whole-cell recording. FK960 (100 nM) significantly increased the amplitude of the EPSP, which was unchanged when changeover was made to control artificial cerebrospinal fluid (aCSF). FK960 had no significant action on membrane potential, input resistance, or the early GABAergic inhibitory postsynaptic current. The decay phase of the excitatory postsynaptic current was not significantly altered by exposure to FK960, indicating that the properties of desensitization and/or deactivation were unchanged and suggesting that the action of FK960 was unlikely to be the result of changes in the properties of the postsynaptic (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors. The quantal content of the EPSP (1/CV(2)) increased after exposure to FK960 but not to control aCSF. Methyllycaconitine or alpha-bungarotoxin blocked the modulatory action of FK960 on the EPSP, and the finding that these alpha 7-nicotinic acetylcholine receptor (alpha 7nAChR) antagonists were effective raises the possibility that FK960 up-regulates the contribution of acetylcholine to synaptic efficacy in the hippocampus. It is concluded that FK960 increases the quantal release of glutamate from Schaffer collateral-commissural nerve terminals in area CA1 of the hippocampus either by changing the ambient level of acetylcholine or by positively modulating the activity of alpha 7nAChRs located on glutamatergic nerve terminals.

[3H]dofetilide binding in SHSY5Y and HEK293 cells expressing a HERG-like K+ channel?

The pharmacological characteristics of [3H]dofetilide binding in SHSY5Y, HEK293 and CHO-K1 cells were examined, and in parallel whole cell recordings used to characterise HERG-like K+ currents. Dofetilide affinity was similar in the human cell lines, SHSY5Y (Kd=99.6 nM) and HEK293 (Kd=102.9 nM), but 10 times lower in CHO-K1 cells (Kd=1200 nM). In contrast, clofilium and E4031 had a similar affinity in all three cell lines, whereas WAY 123,398 had no effect. Electrophysiological studies showed that SHSY5Y cells contained a HERG-like K+ current blocked by application of dofetilide to either side of the membrane. Block was faster when dofetilide was applied intracellularly. In contrast, HEK293 and CHO-K1 cells contained no such current, despite the presence of a partial cDNA for HERG in the former. That [3H]dofetilide is specific for I(Kr)/HERG may be questionable, as HEK293 and CHO-K1 cells contain no such functional K+ current.

Serum withdrawal causes apoptosis in SHSY 5Y cells.

A proportion of differentiated SH-SY5Y cells undergo cell death in response to withdrawal of serum. This death manifests the hallmark features of apoptosis including changes in nuclear morphology, processing and activation of caspase 3 and cleavage of the caspase 3 substrates acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin and poly(ADP-ribose) polymerase. These findings represent the first demonstration of serum withdrawal induced apoptosis in SH-SY5Y cells. The reduction in viability induced by serum deprivation and assessed using an inhibitor of mitochondrial respiration can be partially inhibited by FK506, but FK506 does not prevent caspase 3 processing or cleavage of caspase 3 substrates. FK506 is also able to promote the viability of a small proportion of embryonic mouse sensory neurons following nerve growth factor-withdrawal induced apoptosis. FK506 did not promote viability in either cell type in the absence of serum- or nerve growth factor-withdrawal. These observations are consistent with a survival-promoting effect of FK506 in cultured neurons.

Caspase 6 activity initiates caspase 3 activation in cerebellar granule cell apoptosis.

Using a well documented ex vivo system consisting of rodent cerebellar granule cells (CGCs) the activation of caspases 3 and 6 during apoptosis induced by withdrawal of trophic support was analyzed. At the time of deprivation, the addition of the irreversible, broad-spectrum caspase inhibitor zVADfmk or the cell permeable, caspase 6 inhibitor CP-VEID-cho can transiently suppress the appearance of apoptosis, including the early appearance of DNA fragmentation. Using immunoblotting and fluorogenic peptide assays we observe deprivation-induced activation of caspases 3 and 6, but not caspase 9. Furthermore, active caspase 6 is capable of processing and activating procaspase 3 in cellular extracts prepared from non-apoptotic CGCs, whereas caspase 3 failed to activate caspase 6. In consonant with this, the cell permeable caspase 6 inhibitor prevented deprivation-induced caspase 3 activation whereas a cell permeable caspase 3 inhibitor, CP-DEVD-cho, had no effect on caspase 6 activation. This would indicate that caspase 6 is a significant inducer of the early caspase 3 activity in apoptotic CGCs.

Characterization of 5-HT-sensitive potassium conductances in neonatal rat facial motoneurones in vitro.

1. The properties of the 5-HT-sensitive K+ conductance of neonatal rat facial motoneurones were examined in brainstem slices using whole-cell patch-clamp techniques. 2. In a small proportion of motoneurones, 5-hydroxytryptamine (5-HT) evoked an inward current mediated solely by a decrease in K+ conductance. The reversal potential (V5-HT) was dependent on the external K+ concentration and the 5-HT-evoked current (I5-HT) displayed a linear current-voltage (I-V) relationship. 3. In the remaining motoneurones, the 5-HT-evoked decrease in K+ conductance could only be observed in isolation once a concomitant 5-HT-mediated enhancement of the hyperpolarization-activated current, Ih, had been abolished with the Ih blocker, ZD-7288. 4. External Cs+ also abolished the Ih-mediated component of I5-HT but, in addition, blocked part of the 5-HT-sensitive K+ current. At potentials hyperpolarized to V5-HT, Cs+ voltage dependently blocked I5-HT while at potentials depolarized to V5-HT, I5-HT was largely unaffected. Ba2+ and Rb+ had identical actions to Cs+ on the 5-HT-sensitive K+ current. 5. The Ba2+-, Rb+- and Cs+-sensitive component of the 5-HT-sensitive K+ current inwardly rectified with a reversal potential that was dependent on the K+ equilibrium potential (EK). 6. Replacing external Na+ with N-methyl-D-glucamine, blocking Ca2+ entry, or preventing an increase in intracellular [Ca2+] with BAPTA, all failed to alter I5-HT at potentials depolarized to EK. 7. I5-HT at depolarized potentials was reversibly blocked by 4-aminopyridine (4 mM) but not tetraethylammonium chloride (30 mM) and did not show inactivation during depolarizing voltage pulses (1.5 s duration). 8. The results suggest that, in addition to enhancing Ih, 5-HT modulates two distinct K+ conductances in neonatal rat facial motoneurones. The actions of Cs+, Ba2+ and Rb+ support the involvement of a member of the inwardly rectifying family of K+ channels while the other K+ channel may belong to the voltage-gated family.

Modulation of IH by 5-HT in neonatal rat motoneurones in vitro: mediation through a phosphorylation independent action of cAMP.

The depolarization of adult and neonatal rat facial and spinal motoneurones by 5-hydroxytryptamine (5-HT) in part involves an enhancement of the hyperpolarization-activated, inward-rectifier, IH. Under experimental conditions which promote this action, 5-HT evokes an inward current which can be mimicked by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP) and potentiated by the cAMP-specific phosphodiesterase inhibitor Ro 20-1724. In this study, we show that this action of 5-HT can be blocked by the adenylyl cyclase inhibitors 2'3'-dideoxyadenosine (2',3'-DDA). 5'-adenylimidodiphosphate (AMP-PNP) and SQ-22536 (9-(tetrahydro-2-furyl)adenine), but not by external or internal application of the protein kinase inhibitors H-7, staurosporine and chelerythrine. The most recently cloned 5-HT receptor subtypes, 5-HT4, 5-HT6 and 5-HT7, can all stimulate adenylyl cyclase when activated. In the presence of internal GTP-gamma-S, 5-HT irreversibly enhanced IH. The 5-HT-induced inward current could be reversibly blocked by methysergide, but not by the 5-HT4 receptor antagonist GR-113808A, the 5-HT6 and 5-HT7 antagonist clozapine and the 5-HT1A antagonist WAY-100365. 5-Methoxytryptamine (5-MeOT) and 5-carboxamidotryptamine (5-CT) mimicked the action of 5-HT with a rank order of potency of 5-HT = 5MeOT > 5-CT. Surprisingly, 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH DPAT), a 5-HT1A and 5-HT7 agonist was inactive on facial motoneurones unlike its reported agonist action on spinal motoneurones. It is proposed that cAMP produced by 5-HT-mediated stimulation of adenylyl cyclase acts in a phosphorylation-independent manner, possibly directly, on the IH channel. The 5-HT receptor subtype mediating this response cannot be correlated with any of the classified 5-HT receptor subtypes that stimulate adenylyl cyclase.

Comparison of the patterns of altered cerebral glucose utilisation produced by competitive and non-competitive NMDA receptor antagonists.

Recent studies indicate that competitive and non-competitive NMDA receptor antagonists can be readily distinguished by their effects on local cerebral glucose utilisation (1CGU). In the present study we compare the effects of the novel NMDA antagonist, (+)-1-methyl-1phenyl-1,2,3,4-tetrahydroisoquinoline (FR115427) on 1CGU, comparing its metabolic profile with that of the non-competitive NMDA receptor antagonist, dizocilpine (MK801) and of the competitive NMDA receptor antagonist CGS19755, using the 2-deoxyglucose metabolic mapping approach. Local cerebral glucose utilisation was measured in 80 anatomically discrete regions of the conscious rat brain using [14C]2-deoxyglucose quantitative autoradiography. Studies were initiated 10 min after the administration of FR115427 (0.1-3 mg/kg i.v.; n = 20), dizocilpine (0.03-0.3 mg/kg; n = 15), CGS19755 (1-30 mg/kg; n = 15) or saline (2 ml/kg; n = 5). Dizocilpine produced characteristic alterations in 1CGU with widespread increases in 1CGU in primary olfactory and limbic areas while reducing 1CGU in somatosensory and motor cortex. FR115427 produced a pattern of altered 1CGU which was broadly similar to that elicited by dizocilpine with increases in 1CGU in the pontine nuclei, presubiculum and hippocampus and reductions in somatosensory and motor cortex and within components of the auditory system. However, FR115427 was approximately 30-fold less potent than dizocilpine in this regard. In limbic structures, the effects of FR115427 were less pronounced than those produced by dizocilpine. Increases in 1CGU of 62-98% were found in retrosplenial, piriform and entorhinal cortex of dizocilpine-treated rats whereas these areas appeared relatively unaffected following FR115427 administration. A comparison of the pattern of metabolic response produced by each of these agents was performed by constructing a hierarchy of regional responsiveness using the f statistic: while focal differences in the metabolic profiles of dizocilpine and FR115427 were evident, a plot of the regional f values for dizocilpine and FR115427 revealed a strong overall relationship between the metabolic responses with a Pearson's product moment correlation of 0.78. In contrast, the correlation between the patterns produced by CGS19755 and that for dizocilpine or FR115427 was poor (r = 0.28 and 0.5 respectively).