Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus

The excitatory amino acid neurotransmitter glutamate participates in the control of most (and possibly all) neuroendocrine systems in the hypothalamus. This control is exerted by binding to two classes of membrane receptors, the ionotropic and metabotropic receptor families, which differ in their structure and mechanisms of signal transduction. To gain a better understanding about the precise sites of action of glutamate and the subunit compositions of the receptors involved in the glutamatergic neurotransmission in the hypothalamus and septum, in situ hybridization was used with 35S-labeled cRNA probes for the different ionotropic receptor subunits, including glutamate receptor subunits 1-4 (GluR1-GluR4), kainate-2, GluR5-GluR7, N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), and NMDAR2A-NMDAR2D. The results showed that subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-preferring, kainate-preferring, and NMDA-preferring receptor subunits are distributed widely but heterogeneously and that the GluR1, GluR2, kainate-2, NMDAR1, NMDAR2A, and NMDAR2B subunits are the most abundant in the hypothalamus. Thus, GluR1 subunit mRNA was prominent in the lateral septum, preoptic area, mediobasal hypothalamus, and tuberomammillary nucleus, whereas kainate-2 subunit mRNA was abundant in the medial septum-diagonal band, median and anteroventral preoptic nuclei, and supraoptic nuclei as well as the magnocellular portion of the posterior paraventricular nucleus. Regions that contained the highest levels of NMDAR1 subunit mRNA included the septum, the median preoptic nucleus, the anteroventral periventricular nucleus, and the supraoptic and suprachiasmatic nuclei as well as the arcuate nucleus. Together, the extensive distribution of the different GluR subunit mRNAs strengthen the view that glutamate is a major excitatory neurotransmitter in the hypothalamus. The overlap in the distribution of the various subunit mRNAs suggests that many neurons can express GluR channels that belong to different families, which would allow a differential regulation of the target neurons by glutamate.     

Use of catechol‐O‐methyltransferase inhibition to minimize L‐3,4‐dihydroxyphenylalanine‐induced dyskinesia in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐lesioned macaque

L‐3,4‐dihydroxyphenylalanine (L‐DOPA)‐induced dyskinesia is a complication of dopaminergic treatment in Parkinson's disease. Lowering the L‐DOPA dose reduces dyskinesia but also reduces the antiparkinsonian benefit. A therapy that could enhance the antiparkinsonian action of low‐dose L‐DOPA (LDl) without exacerbating dyskinesia would thus be of considerable therapeutic benefit. This study assessed whether catechol‐O‐methyltransferase (COMT) inhibition, as an add‐on to LDl, might be a means to achieve this goal. Cynomolgus macaques were administered 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. Dyskinesia was established by chronic treatment with L‐DOPA. Two doses of L‐DOPA were identified – high‐dose L‐DOPA (LDh), which provided good antiparkinsonian benefit but was compromised by disabling dyskinesia, and LDl, which was sub‐threshold for providing significant antiparkinsonian benefit, without dyskinesia. LDh and LDl were administered in acute challenges in combination with vehicle and, for LDl, with the COMT inhibitor entacapone (5, 15 and 45 mg/kg). The duration of antiparkinsonian benefit (ON‐time), parkinsonism and dyskinesia were determined. The ON‐time after LDh was ～170 min and the ON‐time after LDl alone (～98 min) was not significantly different to vehicle (～37 min). In combination with LDl, entacapone significantly increased the ON‐time (5, 15 and 45 mg/kg being ～123, ～148 and ～180 min, respectively). The ON‐time after LDl/entacapone 45 mg/kg was not different to that after LDh. However, whereas the percentage ON‐time that was compromised by disabling dyskinesia was ～56% with LDh, it was only ～31% with LDl/entacapone 45 mg/kg. In addition to the well‐recognized action of COMT inhibition to reduce wearing‐OFF, the data presented suggest that COMT inhibition in combination with low doses of L‐DOPA has potential as a strategy to alleviate dyskinesia.     

Learning new sequential stepping patterns requires striatal plasticity during the earliest phase of acquisition

Animals including humans execute motor behavior to reach their goals. For this purpose, they must choose correct strategies according to environmental conditions and shape many parameters of their movements, including their serial order and timing. To investigate the neurobiology underlying such skills, we used a multi‐sensor equipped, motor‐driven running wheel with adjustable sequences of foothold pegs on which mice ran to obtain water reward. When the peg patterns changed from a familiar pattern to a new pattern, the mice had to learn and implement new locomotor strategies in order to receive reward. We found that the accuracy of stepping and the achievement of water reward improved with the new learning after changes in the peg‐pattern, and c‐Fos expression levels assayed after the first post‐switch session were high in both dorsolateral striatum and motor cortex, relative to post‐switch plateau levels. Combined in situ hybridization and immunohistochemistry of striatal sections demonstrated that both enkephalin‐positive (indirect pathway) neurons and substance P‐positive (direct pathway) neurons were recruited specifically after the pattern switches, as were interneurons expressing neuronal nitric oxide synthase. When we blocked N‐methyl‐D‐aspartate (NMDA) receptors in the dorsolateral striatum by injecting the NMDA receptor antagonist, D‐2‐amino‐5‐phosphonopentanoic acid (AP5), we found delays in early post‐switch improvement in performance. These findings suggest that the dorsolateral striatum is activated on detecting shifts in environment to adapt motor behavior to the new context via NMDA‐dependent plasticity, and that this plasticity may underlie forming and breaking skills and habits as well as to behavioral difficulties in clinical disorders.     

Activation of 5-HT2 receptors enhances the release of acetylcholine in the prefrontal cortex and hippocampus of the rat

The role of 5-HT2 receptors in the regulation of acetylcholine (ACh) release was examined in the medial prefrontal cortex and dorsal hippocampus using in vivo microdialysis. The 5-HT(2A/2C) agonist +/-1-(2,5-dimethoxy-4-iodophenyl) -2- aminopropane hydrochloride (DOI) (1 and 2 mg/kg, i.p.) significantly increased the extracellular concentration of ACh in both brain regions, and this response was attenuated in rats treated with the 5-HT(2A/2B/2C) antagonist LY-53,857 (3 mg/kg, i.p.). Treatment with LY-53,857 alone did not significantly alter ACh release in either brain region The 5-HT(2C) agonist 6-chloro-2-(1-piperazinyl)-pyrazine) (MK-212) (5 mg/kg, i.p.) significantly enhanced the release of ACh in both the prefrontal cortex and hippocampus, whereas the 5-HT2 agonist mescaline (10 mg/kg, i.p.) produced a 2-fold increase in ACh release only in the prefrontal cortex. Intracortical, but not intrahippocampal, infusion of DOI (100 microM) significantly enhanced the release of ACh, and intracortical infusion of LY-53,857 (100 microM) significantly attenuated this response. These results suggest that the release of ACh in the prefrontal cortex and hippocampus is influenced by 5-HT2 receptor mechanisms. The increase in release of ACh induced by DOI in the prefrontal cortex, but not in the hippocampus, appears to be due to 5-HT2 receptor mechanisms localized within this brain region. Furthermore, it appears that the prefrontal cortex is more sensitive than the dorsal hippocampus to the stimulatory effect of 5-HT2 agonists on ACh release.     

Acute and chronic effects of citalopram on 5‐HT1A receptor—Labeling by [18F]MPPF and—Coupling to receptors‐G proteins

Selective serotonin reuptake inhibitors take several weeks to produce their maximal therapeutic antidepressant effect. This delay has been attributed to the gradual desensitization of somatodendritic serotonin 5‐HT_1A autoreceptors. We evaluated adaptive changes of 5‐HT_1A receptors after acute and chronic citalopram challenges in rat. Small animal positron emission tomography trial and quantitative ex vivo autoradiography studies using [¹⁸F]MPPF were employed, as well as in vitro 8‐OH‐DPAT‐stimulated [³⁵S]‐GTPγS binding assay. Additionally, 5‐HT_1A receptor knock‐out mice were used to assess the specificity of [¹⁸F]MPPF. Acute treatment with citalopram did not alter [¹⁸F]MPPF binding in dorsal raphe nucleus (DR), frontal cortex, or hippocampus. The absence of [¹⁸F]MPPF binding in the brain of 5‐HT_1A knock‐out mice demonstrates the specificity of MPPF for 5‐HT_1A receptor brain imaging, but the high affinity of [¹⁸F]MPPF compared to 5‐HT suggests that it would only be displaced by dramatic increases in extracellular 5‐HT. Chronic citalopram did not modify 5‐HT_1A receptor density in any of the brain regions studied. In addition, this treatment did not modify 8‐OH‐DPAT‐stimulated [³⁵S]‐GTPγS binding in DR, although a significant increase was observed in frontal cortex and hippocampus. [¹⁸F]MPPF appears to be an efficient radioligand to quantify specifically 5‐HT_1A receptor density in brain imaging. The delayed therapeutic efficacy of citalopram did not appear to be linked to either a downregulation of 5‐HT_1A receptors or to a 5‐HT_1A receptor‐G protein decoupling process in serotonergic neurons, but to increased functional sensitivity of postsynaptic 5‐HT_1A receptors. Synapse 63:106–116, 2009. ©2008 Wiley‐Liss, Inc.     

Kampo therapy as an alternative to pharmacotherapy using antipsychotic medicines for behavioral and psychological symptoms of dementia (BPSD)

The behavioral and psychological symptoms of dementia (BPSD), including aggression, agitation, screaming, wandering, hallucinations, and delusions, occur in 50–90% of patients with dementia, and have a negative impact on the activity of daily living (ADL) of patients, as well as caregivers. Patients with severe BPSD often require management with antipsychotic medicines. However, an increased mortality rate has been reported in patients with dementia taking antipsychotic medicine and, thus, there is an urgent need to develop safer treatments for BPSD. Kampo medicines are an alternative to antipsychotic medicines and several Kampo medicines have been reported to be effective in the treatment of BPSD. Oren‐gedoku‐to has been reported to be effective for the treatment of irritability and sullenness in patients with vascular dementia, as well as improving excitement, depression, anxiety, and restlessness of patients with cerebrovascular lesions. Choto‐san has been reported to be effective in the treatment of delirium, insomnia, and hallucinations/delusions in patients with vascular dementia. Toki‐syakuyaku‐san has been reported to improve emotional lability, restlessness, and sleep disturbances in patients with dementia. Yokukan‐san has been reported to be effective for hallucinations, agitation/aggression, irritability/lability, and aberrant motor activity, as well as being effective in the treatment of visual hallucinations in patients with dementia with Lewy bodies (DLB). A multicenter randomized crossover study confirmed that Yokukan‐san is effective in the treatment of BPSD and is well‐tolerated. Kampo medicines do not induce extrapyramidal or anticholinergic symptoms and have no adverse effects on ADL or cognitive function. Thus, Kampo therapy is recommended for patients who cannot tolerate treatment with neuroleptics, patients who have extrapyramidal symptoms and gait disturbance, and patients with DLB. In future, to confirm the effectiveness of Kampo medicines in the treatment of BPSD, further studies, such as randomized control trials, are needed. In addition, basic studies are required to elucidate the processes by which Kampo medicines are metabolized, as well as any interactions between Western and Kampo medicines.     

Connectivity of cone photoreceptor telodendria in the zebrafish retina

The connectivity amongst photoreceptors is critical to their function, as it underpins lateral inhibition and effective translation of stimuli into neural signals. Despite much work characterizing second‐order interneurons in the outer retina, the synapses directly connecting photoreceptors have often been overlooked. Telodendria are fine processes that connect photoreceptor pedicles. They have been observed in diverse vertebrate groups, yet their roles in vision remain speculative. Here, we visualize telodendria via fluorescent protein expression in photoreceptor subtypes. We characterized short wavelength cone telodendria in adult and larval zebrafish retina. Additionally, in the larval retina, we investigated rod telodendria and UV cone telodendria in mutant and transgenic retinas with altered complements of cone types. In the adult retina, telodendria are twice as abundant and branch almost twice as often on blue cones compared to UV cones. Pedicles of neighboring UV and blue cones typically converge into contiguous pairs, despite the regular spacing of their cell bodies. In contrast to adults, larval UV cone telodendria are more numerous (1.3 times) than blue cone telodendria. UV cone telodendria are not detectably affected by ablation of blue cones, and are reduced twofold in mutant larval retina with few UV cones. We thus saw no evidence that telodendria increase in number in the absence of their typical cellular neighbors. We also found that larval rod telodendria are less abundant than short wavelength cone telodendria. In summary, we describe the development and morphology of zebrafish photoreceptor synaptic connectivity toward appreciating the function of telodendria in visual signal processing.     

Perampanel,a selective,noncompetitive α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor antagonist,as adjunctive therapy for refractory partial‐onset seizures: Interim results from phase III,extension study 307

Purpose: To evaluate safety, tolerability, and seizure outcome data during long‐term treatment with once‐daily adjunctive perampanel (up to 12 mg/day) in patients with refractory partial‐onset seizures. Methods: Study 307 was an extension study for patients completing the double‐blind phase of three pivotal phase III trials (studies 304, 305, and 306). The study consisted of two phases: an open‐label treatment phase (including a 16‐week blinded conversion period and a planned 256‐week maintenance period) and a 4‐week follow‐up phase. Patients were blindly titrated during the conversion period to their individual maximum tolerated dose (maximum 12 mg/day). Adverse events (AEs) were monitored throughout the study and seizure frequency recorded. The interim data cutoff date for analyses was December 1, 2010. Key Findings: In total, 1,218 patients were enrolled in the study. At the interim cutoff date, 1,186 patients were in the safety analysis set; 1,089 (91.8%) patients had >16 weeks of exposure to perampanel, 580 (48.9%) patients had >1 year of exposure, and 19 (1.6%) patients had >2 years of exposure. At the interim analysis, 840 (70.8%) patients remained on perampanel treatment. The large majority of patients (n = 1,084 [91%]) were titrated to 10 mg or 12 mg/day. Median (range) duration of exposure was 51.4 (1.1–128.1) weeks. Treatment‐emergent AEs were reported in 87.4% of patients. The most frequent were dizziness (43.9%), somnolence (20.2%), headache (16.7%), and fatigue (12.1%). Serious AEs were reported in 13.2% of patients. In the intent‐to‐treat analysis set (n = 1,207), the frequency of all seizures decreased over the first 26 weeks of perampanel treatment in patients with at least 26 weeks of exposure to perampanel (n = 1,006 [83.3%]); this reduction was maintained in patients with at least 1 year of exposure (n = 588 [48.7%]). The overall median percent changes in seizure frequency in patients included in each 13‐week interval of perampanel treatment were ?39.2% for weeks 14–26 (n = 1,114), ?46.5% for weeks 40–52 (n = 731), and ?58.1% for weeks 92–104 (n = 59). Overall responder rates in patients included in each 13‐week interval of perampanel treatment were 41.4% for weeks 14–26 (n = 1,114), 46.9% for weeks 40–52 (n = 731), and 62.7% for weeks 92–104 (n = 59). During the blinded conversion period, the reduction in seizure frequency in patients previously randomized to placebo (?42.4%, n = 369) was similar to that in patients previously randomized to perampanel (?41.5%, n = 817). Significance: Consistent with pivotal phase III trials, these interim results demonstrated that perampanel had a favorable tolerability profile in patients with refractory partial‐onset seizures over the longer term. The decrease in seizure frequency was consistent and maintained in those patients over at least 1 year of perampanel exposure.     

Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.     

Development of the Korean version of Alzheimer's Disease Assessment Scale (ADAS-K)

OBJECTIVE: The purpose of this study was the development of the Korean Version of Alzheimer's Disease Assessment Scale (ADAS-K). METHOD: ADAS-K was administrated to 84 AD patients as well as 105 non-demented control subjects. Three aspects of reliability were tested. To evaluate the validity of ADAS-K, discriminant validity and concurrent validity were tested. To evaluate the sensitivity of ADAS-K to disease severity, all subjects, AD patients and control subjects, were grouped by CDR scale and their mean scores on ADAS-K were compared. RESULT: ADAS-K demonstrated high levels of reliability. Mean ADAS-K scores for AD patients were significantly different from the control group (p < 0.01). Furthermore, ADAS-K exhibited significant correlations with other tests and scales (range 0.45-0.85, p < 0.01). In ROC curve analysis, ADAS-K displayed high diagnostic efficacy and the optimal cut-off point was selected between 18/19. ADAS-K was able to discriminate the degree of AD severity according to CDR classification. Our results suggested that ADAS-K-cog was sensitive to very mild AD. CONCLUSION: We demonstrated that ADAS-K is a reliable and valid instrument not only for AD diagnosis but also for evaluation of its severity.     

Perampanel in patients with refractory and super‐refractory status epilepticus in a neurological intensive care unit: A single‐center audit of 30 patients

In refractory status epilepticus (SE), γ‐aminobutyric acidergic drugs become less effective and glutamate plays a major role in seizure perpetuation. Data on the efficacy of perampanel (PER) in treatment of refractory SE in humans are limited. Here, we present a single‐center case series of patients with refractory SE who received PER orally in an intensive care unit. We retrospectively analyzed treatment response, outcome, and adverse effects of all patients with refractory SE in our Neurological Intensive Care Unit who received add‐on PER between September 2012 and February 2018. Thirty patients with refractory SE (median = 72 years, range = 18‐91, 77% women) were included. In 14 patients (47%), a high‐dose approach was used, with a median initial dose of 24 mg (range = 16‐32). In five patients (17%), SE could be terminated after PER administration (median dose = 6 mg, range = 6‐20 mg, 2/5 patients in high‐dose group). Clinical response was observed after a median of 24 hours (range = 8‐48 hours), whereas electroencephalogram resolved after a median of 60 hours (range = 12‐72 hours). Time to treatment response tended to be shorter in patients receiving high‐dose PER (median clinical response = 16 hours vs 18 hours; electroencephalographic response = 24 hours vs 72 hours), but groups were too small for statistical analysis. Continuous cardiorespiratory monitoring showed no changes in cardiorespiratory function after “standard” and “high‐dose” treatment. Elevated liver enzymes without clinical symptoms were observed after a median of 6 days in seven of 30 patients (23%; 57% high dose vs 43% standard dose), of whom six also received treatment with phenytoin (PHT). Outcome was unfavorable (death, persistent vegetative state) in 13 patients (43%; 39% high dose vs 61% standard dose), and good recovery (no significant disability, moderate disability) was achieved in nine patients (56% high dose vs 44% standard dose). Oral PER in loading doses up to 32 mg were well tolerated but could terminate SE only in a few patients (5/30; 17%). Long duration of SE, route of administration, and severe underlying brain dysfunction might be responsible for the modest result. An intravenous formulation is highly desired to explore the full clinical utility in the treatment of refractory SE.     

Diversity in anti-N-methyl-D-aspartate receptor encephalitis: case-based evidence

Antibodies against N-methyl-D-aspartate receptor (NMDAR) are identified in the form of immune-mediated encephalitis in which typical manifestations include neuropsychiatric symptoms, seizures, abnormal movements, dysautonomia and hypoventilation. The authors report two cases of anti-NMDAR encephalitis with different presentations and patterns of progression. The first patient presented with status epilepticus and later developed psychosis, pyramidal signs and diffuse encephalopathy. The second patient presented with acute psychosis followed a week later by seizures, dystonia, rigidity, oromandibular dyskinesias and dysautonomia. Possible mechanisms responsible for the clinical manifestations of this disease are discussed in light of recently described additional clinical and laboratory findings.     

An autopsied case of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease presenting with hyperintensities on diffusion‐weighted MRI before clinical onset

A 78‐year‐old Japanese man presented with rapidly progressive dementia and gait disturbances. Eight months before the onset of clinical symptoms, diffusion‐weighted magnetic resonance imaging (DWI) demonstrated hyperintensities in the right temporal, right parietal and left medial occipital cortices. Two weeks after symptom onset, DWI showed extensive hyperintensity in the bilateral cerebral cortex, with regions of higher brightness that existed prior to symptom onset still present. Four weeks after clinical onset, periodic sharp wave complexes were identified on an electroencephalogram. Myoclonus was observed 8 weeks after clinical onset. The patient reached an akinetic mutism state and died 5 months after onset. Neuropathological examination showed widespread cerebral neocortical involvement of fine vacuole‐type spongiform changes with large confluent vacuole‐type spongiform changes. Spongiform degeneration with neuron loss and hypertrophic astrocytosis was also observed in the striatum and medial thalamus. The inferior olivary nucleus showed severe neuron loss with hypertrophic astrocytosis. Prion protein (PrP) immunostaining showed widespread synaptic‐type PrP deposition with perivacuolar‐type PrP deposition in the cerebral neocortex. Mild to moderate PrP deposition was also observed extensively in the basal ganglia, thalamus, cerebellum and brainstem, but it was not apparent in the inferior olivary nucleus. PrP gene analysis showed no mutations, and polymorphic codon 129 showed methionine homozygosity. Western blot analysis of protease‐resistant PrP showed both type 1 scrapie type PrP (PrP^Sc) and type 2 PrP^Sc. Based on the relationship between the neuroimaging and pathological findings, we speculated that cerebral cortical lesions with large confluent vacuoles and type 2 PrP^Sc would show higher brightness and continuous hyperintensity on DWI than those with fine vacuoles and type 1 PrP^Sc. We believe the present patient had a combined form of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease (sCJD), which suggests a broader spectrum of sCJD clinicopathological findings.     

The comparison of mouse full metallothionein‐1 versus α and β domains and metallothionein‐1‐to‐3 mutation following traumatic brain injury reveals different biological motifs

Traumatic injury to the brain is one of the leading causes of injury‐related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock‐out mice. We herewith report the results comparing full mouse MT‐1 with the independent α and β domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota‐rod tests; all the proteins showed a modest effect in the former test, while only full MT‐1 improved the performance of animals in the rota‐rod, and the α domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host‐response genes such as GFAP, Mac1 and ICAM, in some cases being the β domain more effective than the α domain or even the full MT‐1. A MT‐1‐to‐MT‐3 mutation blunted some but not all the effects caused by the normal MT‐1, and in some cases increased its potency. Thus, splitting the two MT‐1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions. © 2010 Wiley‐Liss, Inc.