Enkephalin Peptides Research Articles

Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations

Macrocyclic glycopeptide based liquid chromatography stationary phases are known for their highly selective peptide separations. Fast and ultrafast (t R < 1min) high-efficiency separations were achieved with superficially porous particle (SPP)-based stationary phases. Separations of pharmaceutically important classes of peptides such as enkephalins and bradykinins have been achieved in less than 5min in isocratic elution modes. Selectivity for peptides structurally similar to one another was increased with use of teicoplanin-based stationary phases compared with commercial C18 stationary phases. Ultrafast isocratic separations of structurally related peptides were achieved with teicoplanin- and vancomycin-based short SPP columns. Acidic mobile phases produced better separations. Ammonium formate was the optimal mobile phase buffer additive. Use of an appropriate combination of a macrocyclic glycopeptide stationary phase and a mobile phase permits faster and more electrospray ionization mass spectrometry compatible isocratic separations than previous gradient approaches. The tryptic peptide separation characteristics of the teicoplanin stationary phase are demonstrated. Additionally, compared with commercial C18 stationary phases, teicoplanin showed tryptic peptide separations with different selectivities. Graphical Abstract Ultrafast separation of enkephalin peptide epimers.

Reduced thermal sensitivity and increased opioidergic tone in the TASTPM mouse model of Alzheimer's disease.

Individuals with Alzheimer's disease (AD) are in susceptible patient groups in which pain is an important clinical issue that is often underdiagnosed. However, it is unclear whether decreased pain complaints in patients with AD result from elevated pain tolerance or an impaired ability to communicate sensations. Here, we explored if AD-related pathology is present in key regions of the pain pathway and assessed whether nociceptive thresholds to acute noxious stimulation are altered in the double-mutant APPswe × PS1.M146V (TASTPM) transgenic mouse model of AD. TASTPM mice exhibited an age-dependant cognitive deficit at the age of 6 months, but not at 4 months, a deficit that was accompanied by amyloid plaques in the cortex, hippocampus, and thalamus. In the spinal cord, β-amyloid (APP/Aβ) immunoreactivity was observed in dorsal and ventral horn neurons, and the expression of vesicular glutamate transporter 2 (VGLUT2) was significantly reduced, while the expression of the inhibitory peptides enkephalins was increased in TASTPM dorsal horn, consistent with an increased inhibitory tone. TASTPM mice displayed reduced sensitivity to acute noxious heat, which was reversed by naloxone, an opioid antagonist. This study suggests that increased inhibition and decreased excitation in the spinal cord may be responsible for the reduced thermal sensitivity associated with AD-related pathology.

Photoinduced fragmentation of gas-phase protonated leucine- enkephalin peptide in the VUV range

In this article we report new results for action spectroscopy of protonated peptide Leucine enkephalin (YGGFL). By coupling a linear ion trap mass spectrometer with a vacuum ultraviolet (VUV) synchrotron radiation beamline, we investigate photofragmentation pattern of this peptide, through the analysis of tandem mass spectra recorded over a range of VUV photon energies, below and above the ionization energy. The obtained fragmentation patterns are discussed and compared to previous results.

SY03-4THE ENDOGENOUS OPIOID SYSTEM: DYSREGULATION IN THE STRIATUM OF HUMAN ALCOHOLICS

The endogenous opioid peptides dynorphins and enkephalins may be involved in brain-area specific synaptic adaptations relevant for different stages of an addiction cycle. We compared the levels of prodynorphin ( PDYN ) and proenkephalin ( PENK ) mRNAs (by qRT-PCR), and dynorphins and enkephalins (by radioimmunoassay) in the caudate …

A newly identified mouse hypothalamic area having bidirectional neural connections with the lateral septum: the perifornical area of the anterior hypothalamus rich in chondroitin sulfate proteoglycans.

While previous studies and brain atlases divide the hypothalamus into many nuclei and areas, uncharacterised regions remain. Here, we report a new region in the mouse anterior hypothalamus (AH), a triangular-shaped perifornical area of the anterior hypothalamus (PeFAH) between the paraventricular hypothalamic nucleus and fornix, that abundantly expresses chondroitin sulfate proteoglycans (CSPGs). The PeFAH strongly stained with markers for chondroitin sulfate/CSPGs such as Wisteria floribunda agglutinin and antibodies against aggrecan and chondroitin 6 sulfate. Nissl-stained sections of the PeFAH clearly distinguished it as a region of comparatively low density compared to neighboring regions, the paraventricular nucleus and central division of the anterior hypothalamic area. Immunohistochemical and DNA microarray analyses suggested that PeFAH contains sparsely distributed calretinin-positive neurons and a compact cluster of enkephalinergic neurons. Neuronal tract tracing revealed that both enkephalin- and calretinin-positive neurons project to the lateral septum (LS), while the PeFAH receives input from calbindin-positive LS neurons. These results suggest bidirectional connections between the PeFAH and LS. Considering neuronal subtype and projection, part of PeFAH that includes a cluster of enkephalinergic neurons is similar to the rat perifornical nucleus and guinea pig magnocellular dorsal nucleus. Finally, we examined c-Fos expression after several types of stimuli and found that PeFAH neuronal activity was increased by psychological but not homeostatic stressors. These findings suggest that the PeFAH is a source of enkephalin peptides in the LS and indicate that bidirectional neural connections between these regions may participate in controlling responses to psychological stressors.

Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity

Obesity is an increasingly prevalent disease regulated by genetic and environmental factors. Emerging studies indicate that immune cells, including monocytes, granulocytes and lymphocytes, regulate metabolic homeostasis and are dysregulated in obesity. Group 2 innate lymphoid cells (ILC2s) can regulate adaptive immunity and eosinophil and alternatively activated macrophage responses, and were recently identified in murine white adipose tissue (WAT) where they may act to limit the development of obesity. However, ILC2s have not been identified in human adipose tissue, and the mechanisms by which ILC2s regulate metabolic homeostasis remain unknown. Here we identify ILC2s in human WAT and demonstrate that decreased ILC2 responses in WAT are a conserved characteristic of obesity in humans and mice. Interleukin (IL)-33 was found to be critical for the maintenance of ILC2s in WAT and in limiting adiposity in mice by increasing caloric expenditure. This was associated with recruitment of uncoupling protein 1 (UCP1)(+) beige adipocytes in WAT, a process known as beiging or browning that regulates caloric expenditure. IL-33-induced beiging was dependent on ILC2s, and IL-33 treatment or transfer of IL-33-elicited ILC2s was sufficient to drive beiging independently of the adaptive immune system, eosinophils or IL-4 receptor signalling. We found that ILC2s produce methionine-enkephalin peptides that can act directly on adipocytes to upregulate Ucp1 expression in vitro and that promote beiging in vivo. Collectively, these studies indicate that, in addition to responding to infection or tissue damage, ILC2s can regulate adipose function and metabolic homeostasis in part via production of enkephalin peptides that elicit beiging.

Anti-inflammatory and antinociceptive action of the dimeric enkephalin peptide biphalin in the mouse model of colitis: New potential treatment of abdominal pain associated with inflammatory bowel diseases

Biphalin, a mixed MOP/DOP agonist, displays a potent antinociceptive activity in numerous animal models of pain. The aim of the study was to characterize the anti-inflammatory and antinociceptive action of biphalin in the mouse models of colitis. The anti-inflammatory effect of biphalin (5mg/kg, twice daily, i.c. and i.p.) was characterized in a semi-chronic mouse model of colitis, induced by i.c. injection of trinitrobenzenesulfonic acid (TNBS). The antinociceptive action of biphalin (5mg/kg, i.p. and i.c.) in inflamed mice was assessed in mustard oil-induced model of visceral pain and in the hot plate test. In the semi-chronic mouse model of colitis, biphalin i.c. (5mg/kg), but not i.p. improved colitis macroscopic score (2.88±0.19 and 4.99±0.80 units for biphalin and vehicle treated animals, respectively). Biphalin injected i.p. and i.c. (5mg/kg) displayed a potent antinociceptive action in the mustard oil-induced pain test. In the hot plate test, biphalin (5mg/kg, i.p.) produced a potent antinociceptive activity in inflamed mice, suggesting central site of action. Our data suggest that biphalin may become a novel opioid-based analgesic agent in IBD therapy and warrant further investigation of its pharmacological profile.

Enkephalin knockout male mice are resistant to chronic mild stress.

Enhanced stress reactivity or sensitivity to chronic stress increases the susceptibility to mood pathologies such as major depression. The opioid peptide enkephalin is an important modulator of the stress response. Previous studies using preproenkephalin knockout (PENK KO) mice showed that these animals exhibit abnormal stress reactivity and show increased anxiety behavior in acute stress situations. However, the consequence of enkephalin deficiency in the reactivity to chronic stress conditions is not known. In this study, we therefore submitted wild-type (WT) and PENK KO male mice to chronic stress conditions, using the chronic mild stress (CMS) protocol. Subsequently, we studied the CMS effects on the behavioral and hormonal level and also performed gene expression analyses. In WT animals, CMS increased the expression of the enkephalin gene in the paraventricular nucleus (PVN) of the hypothalamus and elevated the corticosterone levels. In addition, WT mice exhibited enhanced anxiety in the zero-maze test and depression-related behaviors in the sucrose preference and forced swim tests. Surprisingly, in PENK KO mice, we did not detect anxiety and depression-related behavioral changes after the CMS procedure, and even measured a decreased hormonal stress response. These results indicate that PENK KO mice are resistant to the CMS effects, suggesting that enkephalin enhances the reactivity to chronic stress.

Differentiating Sulfopeptide and Phosphopeptide Ions via Resonant Infrared Photodissociation

The post-translational modifications sulfation and phosphorylation pose special challenges to mass spectral analysis due to their isobaric nature and their lability in the gas phase, as both types of peptides dissociate through similar channels upon collisional activation. Here, we present resonant infrared photodissociation based on diagnostic sulfate and phosphate OH stretches, as a means to differentiate sulfated from phosphorylated peptides within the framework of a mass spectrometry platform. The approach is demonstrated for a number of tyrosine-containing peptides, ranging from dipeptides (YG, pYG, and sYG) over tripeptides (GYR, GpYR, and GsYR), to more biologically relevant enkephalin peptides (YGGFL, pYGGFL, and sYGGFL). In all cases, the diagnostic ranges for sulfate OH stretches are established as 3580-3600 cm(-1) and can thus be distinguished from other characteristic hydrogen stretches, such as carboxylic acid OH, alcohol OH, and phosphate OH stretches.

The effect of striatal pre-enkephalin overexpression in the basal ganglia of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.

The midbrain dopamine (DA) cell death underlying Parkinson's disease (PD) is associated with upregulation of pre-enkephalin (pENK) in striatopallidal neurons. Our previous results obtained with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian monkeys suggest that increased striatal expression of pENK mRNA is a compensatory mechanism to alleviate PD-related motor symptoms. In this study, we tested the hypothesis that increased pENK expression in the striatum protects against the neurotoxic insults of MPTP in mice. To this end, recombinant adeno-associated virus serotype 2 also containing green fluorescent protein was used to overexpress pENK prior to DA depletion. Our results showed that overexpression of pENK in the striatum of MPTP mice induced: (i) increased levels of the opioid peptide enkephalin (ENK) in the striatum; (ii) higher densities of ENK-positive fibers in both the globus pallidus (GP) and the substantia nigra; (iii) higher locomotor activity; and (iv) a higher density of striatal tyrosine hydroxylase-positive fibers in the striatum. In addition, striatal overexpression of pENK in MPTP -treated mice led to 52 and 43% higher DA concentrations and DA turnover, respectively, in the GP compared to sham-treated MPTP mice. These observations are in agreement with the idea that increased expression of pENK at an early stage of disease can improve PD symptoms.

A drug delivery strategy: binding enkephalin to asialoglycoprotein receptor by enzymatic galactosylation.

Glycosylation of biopharmaceuticals can mediate cell specific delivery by targeting carbohydrate receptors. Additionally, glycosylation can improve the physico-chemical (drug-like) properties of peptide based drug candidates. The main purpose of this study was to examine if glycosylation of the peptide enkephalin could facilitate its binding to the carbohydrate receptor, asialoglycoprotein. Firstly, we described the one-pot enzymatic galactosylation of lactose modified enkephalin in the presence of uridine-5′-diphosphogalactose 4-epimerase and lipopolysaccharyl α-1,4-galactosyltransferase. Stability experiments using human plasma and Caco-2 cell homogenates showed that glycosylation considerably improved the stability of enkephalin (at least 60% remained stable after a 2 hr incubation at 37°C). In vitro permeability experiments using Caco-2 cells revealed that the permeability of mono- and trisaccharide conjugated enkephalins was 14 and 28 times higher, respectively, than that of enkephalin alone (Papp 3.1×10−8 cm/s). By the methods of surface plasmon resonance and molecular modeling, we demonstrated that the enzymatic glycosylation of enkephalin enabled binding the asialoglycoprotein receptor. The addition of a trisaccharide moiety to enkephalin improved the binding of enkephalin to the asialoglycoprotein receptor two fold (KD = 91 µM). The docking scores from molecular modeling showed that the binding modes and affinities of the glycosylated enkephalin derivatives to the asialoglycoprotein receptor complemented the results from the surface plasmon resonance experiments.

Striatal Pre-Enkephalin Overexpression Improves Huntington’s Disease Symptoms in the R6/2 Mouse Model of Huntington’s Disease

The reduction of pre-enkephalin (pENK) mRNA expression might be an early sign of striatal neuronal dysfunction in Huntington’s disease (HD), due to mutated huntingtin protein. Indeed, striatopallidal (pENK-containing) neurodegeneration occurs at earlier stage of the disease, compare to the loss of striatonigral neurons. However, no data are available about the functional role of striatal pENK in HD. According to the neuroprotective properties of opioids that have been recognized recently, the objective of this study was to investigate whether striatal overexpression of pENK at early stage of HD can improve motor dysfunction, and/or reduce striatal neuronal loss in the R6/2 transgenic mouse model of HD. To achieve this goal recombinant adeno-associated-virus (rAAV2)-containing green fluorescence protein (GFP)-pENK was injected bilaterally in the striatum of R6/2 mice at 5 weeks old to overexpress opioid peptide pENK. Striatal injection of rAAV2-GFP was used as a control. Different behavioral tests were carried out before and/or after striatal injections of rAAV2. The animals were euthanized at 10 weeks old. Our results demonstrate that striatal overexpression of pENK had beneficial effects on behavioral symptoms of HD in R6/2 by: delaying the onset of decline in muscular force; reduction of clasping; improvement of fast motor activity, short-term memory and recognition; as well as normalization of anxiety-like behavior. The improvement of behavioral dysfunction in R6/2 mice having received rAAV2-GFP-pENK associated with upregulation of striatal pENK mRNA; the increased level of enkephalin peptide in the striatum, globus pallidus and substantia nigra; as well as the slight increase in the number of striatal neurons compared with other groups of R6/2. Accordingly, we suggest that at early stage of HD upregulation of striatal enkephalin might play a key role at attenuating illness symptoms.

Neurochemical codes of sympathetic preganglionic neurons activated by glucoprivation

Glucoprivation or hypoglycemia induces a range of counterregulatory responses, including glucose mobilization, reduced glucose utilization, and de novo glucose synthesis. These responses are mediated in part by the sympathetic nervous system. The aim of this study was to determine the chemical codes of sympathetic preganglionic neurons (SPN) activated by glucoprivation, induced by 2-deoxy-D-glucose (2DG). SPN controlling the adrenal glands and celiac ganglia, which ultimately can innervate the liver and pancreas, were targeted together with the superior cervical ganglia (control). 23.9% ± 1.3% of SPN in the T4-T11 region contained c-Fos immunoreactivity following 2DG; 70.3% ± 1.8% of SPN innervating the adrenal glands and 37.4% ± 3% of SPN innervating celiac ganglia were activated. 14.8% ± 3.5% of SPN (C8-T3) innervating superior cervical ganglia were activated. In the C8-T3 region 55% ± 10% of SPN activated contained PPCART, with only 12% ± 3% expressing PPE mRNA, whereas, in the T4-T11 region, 78% ± 4% contained PPE, with only 6.0% ± 0.6% expressing PPCART mRNA. Thus CART is not involved in glucose mobilization. Two chemically distinct populations of SPN (PPE⁺ 57.4% ± 5%, PPE⁻ ∼40%) were identified to regulate adrenaline release in response to glucoprivation. Multiple chemically distinct SPN populations innervating a specific target could suggest their graded recruitment. The two distinct populations of SPN (PPE⁺ 67.6% ± 9%, PPE⁻ ∼30%) projecting to celiac ganglia activated by glucoprivation could direct pancreatic and hepatic or other counterregulatory responses. Nearly all SPN that expressed PPE mRNA and projected to the adrenal glands or celiac ganglia were activated, suggesting a role for the inhibitory peptide enkephalin in responses evoked by glucoprivation.

Mammal retinal distribution of ENKergic amacrine cells and their neurochemical features: Evidence from the PPE-GFP transgenic mice

The neuroactive peptide enkephalin (ENK) has been postulated to play important roles in modulating visual information. The retinal presence of ENKergic cells has been revealed with conventional morphological protocols targeting ENK molecule especially in avian, however, the detailed distribution of ENKergic cells and their specific neurochemical features in the mammal retina remain unclear because of the difficulties in visualizing ENKergic cells efficiently and reliably. To address this question, we took advantage of the preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mice previously generated and identified in our group, and identified the neurochemical characteristics of retinal ENKergic cells. The majority of ENKergic cells occupied the proximal inner nuclear layer with a few displaced in the ganglion cell layer. Further double labeling revealed that most of these ENKergic amacrine cells used inhibitory glycine or gamma-aminobutyric acid as the primary neurotransmitter. However, some of them also utilized excitatory glutamate as the primary neurotransmitter. The present findings suggest that the retinal ENKergic cells fall into a subpopulation of amacrine cells and show predominantly inhibitory as well as less dominantly excitatory neurochemical features. Our findings offered comprehensive morphological evidence for the function of ENKergic amacrine cells of mammal species.

Quantification of enkephalinergic immunoreactivity in the periaqueductal gray of an animal model of endometriosis

Endometriosis is a debilitating disease characterized by pelvic pain, dysmenorrhea and dyspareunia. These symptoms produce stress on the individual and impact daily life activities. It is still poorly understood how stress affects endometriosis associated pain. We aimed to measure levels of the endogenous opioid peptide enkephalin (ENK) in the periaqueductal gray (PAG) of an animal model of endometriosis since PAG plays a critical role in descending modulation of pain via opioidergic mechanisms. Four groups of female rats received either: sham surgery, or surgical endometriosis then no‐stress, controllable or uncontrollable swim stress during 10 consecutive days. Sixty days after surgery, rats were killed and perfused. The brains were removed and sections containing the PAG processed for quantitative light immunohistochemistry for ENK. Results showed that uncontrollable stress exacerbated the vesicle grade in animals with endometriosis as compared to no‐stress or controllable stress. Within the PAG, ENK was mostly observed in the dorsal and lateral regions thru the rostral‐caudal span. Optical densitometry quantification revealed no differences between groups in ENK levels suggesting that neither stress nor endometriosis is affecting ENK levels in the PAG. Future experiments will elucidate whether other opioid peptides or their receptors alter in response to stress and endometriosis. R15AT006373

CNS penetration of the opioid glycopeptide MMP-2200: A microdialysis study

Endogenous opioid peptides enkephalin and dynorphin are major co-transmitters of striatofugal pathways of the basal ganglia. They are involved in the genesis of levodopa-induced dyskinesia and in the modulation of direct and indirect striatal output pathways that are disrupted in Parkinson's disease. One pharmacologic approach is to develop synthetic glycopeptides closely resembling endogenous peptides to restore their normal functions. Glycosylation promotes penetration of the blood–brain barrier. We investigated CNS penetration of the opioid glycopeptide MMP-2200, a mixed δ/μ-agonist based on leu-enkephalin, as measured by in vivo microdialysis and subsequent mass spectrometric analysis in awake, freely moving rats. The glycopeptide (10mg/kg) reaches the dorsolateral striatum (DLS) rapidly after systemic (i.p.) administration and is stably detectable for the duration of the experiment (80min). The detected level at the end of the experiment (around 250pM) is about 10-fold higher than the level of the endogenous leu-enkephalin, measured simultaneously. This is one of the first studies to directly prove that glycosylation of an endogenous opioid peptide leads to excellent blood–brain barrier penetration after systemic injection, and explains robust behavioral effects seen in previous studies by measuring how much glycopeptide reaches the target structure, in this case the DLS.

Disturbances in behavior and cortical enkephalin gene expression during the anticipation of ethanol in rats characterized as high drinkers

The process of ethanol anticipation is a particularly important phenomenon that can determine subsequent drug-taking behavior. Recent studies suggest that systems within the medial prefrontal cortex (mPFC), during anticipation, may contribute to the goal-directed seeking of ethanol. The current investigation examined the possibility that the opioid peptide enkephalin (ENK), known to mediate some of the reinforcing properties of ethanol, may function in the mPFC during the anticipation of ethanol access. Using a limited access (3 h/d) paradigm for 10 days with 20% ethanol, Sprague-Dawley rats were first identified either as low drinkers (LD, <1.0 g/kg/3 h) or as high drinkers (HD, >2.0 g/kg/3 h) that exhibited a long-term phenotype of high ethanol consumption and a significant ethanol deprivation effect. During the anticipation period immediately preceding daily ethanol access, the HD rats compared to LD or Control animals with ad libitum ethanol access exhibited increased anticipatory behaviors, including greater exploratory behavior in a novel open field as revealed by significantly more time spent in the rearing position (+53–65%, p < 0.05) and increased number of rears made (+33–44%, p < 0.05) and greater novelty-seeking behavior in a hole-board apparatus revealed by an increase in total (+50–52%, p < 0.05) and novel nose pokes (+45–48%, p < 0.05). In the HD rats, analysis of the mPFC using real-time quantitative PCR showed significantly greater mRNA levels of ENK (p < 0.05) and the mu-opioid receptor (MOR) (p < 0.05), but not delta-opioid receptor (DOR), and this increase in ENK expression was found, using in situ hybridization, to occur specifically in the prelimbic (PrL) subregion of the mPFC. When injected into the PrL during the anticipation period, a MOR agonist but not DOR agonist significantly increased consumption of 20% ethanol (p < 0.05). These findings support the role of ENK, acting through MOR within the PrL to promote the anticipation and excessive consumption of ethanol.

The prohormone proenkephalin possesses differential conformational features of subdomains revealed by rapid H‐D exchange mass spectrometry

Active enkephalin and related peptide hormones or neurotransmitters are generated by proteolytic processing of inactive prohormone precursors. Little is known about the relative accessibilities of prohormone cleavage sites and conformations of subdomains that undergo proteolytic processing. Therefore, this study investigated the conformational features of the prohormone proenkephalin (PE) by rapid hydrogen-deuterium exchange mass spectrometry (DXMS). DXMS analyzes rates of hydrogen exchange of the polypeptide backbone of PE with deuterium from D(2) O (heavy water) by mass spectrometry, accomplished at sub-second and multisecond time periods. Results showed differential accessibilities of cleavage sites and adjacent subdomains of PE to the aqueous environment. Importantly, protease cleavage sites of PE with greater relative accessibilities correspond to sites most readily cleaved by processing proteases to generate active peptide neurotransmitters. For comparison, peptides derived from PE (by pepsin digestion) displayed greater accessibility to the solvent environment, illustrated by their higher rates of H-D exchange compared to that of intact PE protein. The more limited H-D exchange accessibilities of PE protein, compared to peptides derived from PE, indicate that PE possesses tertiary conformation. These results demonstrate that differential tertiary conformations of PE subdomains undergo ordered proteolytic processing to generate active enkephalin peptides for cell-cell communication in the nervous and endocrine systems.

Spatial and Temporal Distribution Patterns of Enkephalinergic Neurons in Adult and Developing Retinas of the Preproenkephalin-Green Fluorescent Protein Transgenic Mouse

Enkephalin (ENK) peptides are present in the retina of several vertebrate species and play a crucial role in establishing specific circuits during retinal development. However, there is no information available concerning the development of ENKergic neurons in the mouse retina. To address this question, we used preproenkephalin-enhanced green fluorescent protein (GFP) transgenic mice, in which ENKergic neurons are revealed by GFP. Our results showed that most GFP-positive cells were located in the proximal part of the inner nuclear layer with a scattering of GFP-immunoreactive cells in the ganglion cell layer (GCL) in the adult retina. Double immunostaining with syntaxin indicates that GFP expression was restricted to a population of amacrine cells. The proportions of glycine transporter-1 and γ-aminobutyric acid-positive cells among ENKergic neurons were 57.3 ± 2.4% and 10.1 ± 1.8%, respectively. We then injected retrograde tracer into the superior colliculus and observed that none of the ENKergic neurons in the GCL were retrogradely labeled with the tracer. GFP-positive cells were first observed at embryonic day (E) 15 in the inner neuroblastic layer at only very low levels, which gradually increased until E18. After birth, there was a steep rise in GFP expression levels, reaching maximal activity by postnatal day (P) 7. The distribution and intensity of GFP-positive cells at P15 were similar to those of adult retina. It was found that immunoreactive processes in the inner plexiform layer formed strongly stained patches. The present results provide detailed morphological evidence of the cell type and spatial and temporal distribution of ENKergic neurons in the retina.

Multiple Replica Repulsion Technique for Efficient Conformational Sampling of Biological Systems

Here, we propose a technique for sampling complex molecular systems with many degrees of freedom. The technique, termed “multiple replica repulsion” (MRR), does not suffer from poor scaling with the number of degrees of freedom associated with common replica exchange procedures and does not require sampling at high temperatures. The algorithm involves creation of multiple copies (replicas) of the system, which interact with one another through a repulsive potential that can be applied to the system as a whole or to portions of it. The proposed scheme prevents oversampling of the most populated states and provides accurate descriptions of conformational perturbations typically associated with sampling ground-state energy wells. The performance of MRR is illustrated for three systems of increasing complexity. A two-dimensional toy potential surface is used to probe the sampling efficiency as a function of key parameters of the procedure. MRR simulations of the Met-enkephalin pentapeptide, and the 76-residue protein ubiquitin, performed in presence of explicit water molecules and totaling 32 ns each, investigate the ability of MRR to characterize the conformational landscape of the peptide, and the protein native basin, respectively. Results obtained for the enkephalin peptide reflect more closely the extensive conformational flexibility of this peptide than previously reported simulations. Those obtained for ubiquitin show that conformational ensembles sampled by MRR largely encompass structural fluctuations relevant to biological recognition, which occur on the microsecond timescale, or are observed in crystal structures of ubiquitin complexes with other proteins. MRR thus emerges as a very promising simple and versatile technique for modeling the structural plasticity of complex biological systems.