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Spinocerebellar ataxias (SCAs), formerly known as autosomal dominant cerebellar ataxia, are a group of hereditary heterogeneous neurodegenerative disease that contains many subtypes. Spinocerebellar ataxia type 23 (SCA23), one type of SCAs, is caused by mutant prodynorphin (PDYN) gene. A 22-year-old patient was diagnosed with sporadic SCA23 due to gene detection, with a novel identified mutation, PDYN c.647C>T (p.P216L). Located in the dynorphin A-coding-region of PDYN gene, the pathogenic mechanism of the mutation may be relevant to the pathological changes caused by the variant including neurological dysfunction and death of cells. Mild improvement with the patient has been witnessed after active balance and speaking exercise.
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Pain management is an all-time challenge in dentistry. Discontent to pain management is a concern among patients and professionals. Unrelieved pain affects physical and mental well-being contributing to delayed recovery, psychological distress and anxiety. Studies have revealed that chronic pain interferes with normal daily chores of the individual like exercise, sleep, social life and lifestyle. At one end of pain management spectrum are Non-steroidal anti-inflammatory drugs (NSAIDs) while at the other end are the opioids. These drugs are not without constituent side effects. The quest is for new analgesics with potent and long term analgesia with minimal or no side effects. An analgesic that is intermediate in this spectrum is the need of the hour. Opiorphin is an endogenous peptide isolated from human saliva. Opiorphin produces analgesia, by inhibiting enkephalin (ENK) metabolizing enzymes, thus increasing the half-life of circulating ENKs. Apart from being a potent analgesic it can also be a potential biomarker for various systemic and psychosocial disorders. This review focuses on the pharmacological effects of opiorphin and its potential role as a biomarker in various disease conditions.
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Antecedentes. Los efectos deletéreos de suprimir la oxigenación de los tejidos se pueden eliminar con protección celular, utilizando alimentos como el pescado, que contiene litio, ácidos grasos, omega 3, frutas y verduras para aportar antioxidantes. La regeneración celular y tisular se puede mediar por encefalinas. Su acción puede asociarse al ejercicio físico intenso y el trabajo excéntrico encargados de retirar los tejidos lesionados. Objetivo. Conocer algunas experiencias que utilizan la inmersión en piscina para suprimir el dolor y dar calidad de vida al paciente. Materiales y métodos. Se analiza la interacción de diez variables utilizadas como herramientas de trabajo en ocho pacientes escogidos al azar. Resultados. El trabajo excéntrico es la más importante de las herramientas. El daño muscular produce factor de crecimiento muscular y nervioso, lo cual, junto al empleo de insulinas, regenera en conjunto los tejidos. La herramienta de los endocanabinoides participa en la relajación, protección, alimentación y re-funcionalización de los tejidos, a la par que interactúa con las endorfinas para mediar la reparación en el sistema endotelial. Conclusiones. Las imágenes funcionales avanzadas (3 Teslas) de resonancia serán de utilidad para observar y evaluar las interacciones de las herramientas.
Background. Cell protection removes the deleterious effects of suppressing tissue oxygenation by using foods such as fish, which contains lithium, fatty acids, omega 3, fruits and vegetables that provide antioxidants. Enkephalins mediate cell and tissue regeneration. Strenuous exercise and eccentric work are related to enkephalins action in charge of removing the damaged tissues. Objective. To present some professional experiences that use the pool immersion to suppress the pain and to improve the life quality of the patient. Materials and methods. Analysis of ten variables interaction, used as working tools in eight randomly selected patients. Results. Eccentric work is the most important tool. Muscle damage produces nervous and muscle growth factor which, by the use of insulins regenerate tissue altogether. The variable endocannabinoids participates in relaxation, protection, feeding and re-functionalization of tissues; it also interacts with endorphins to mediate the repair in the endothelial system. Conclusions. We conclude that advanced functional imaging (3 Tesla) with resonance would be useful to observe and evaluate the interactions of the variables.
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Objective This study assesses the influence of rizatriptan on calcitonin gene-related peptide (CGRP),proenkephalin (PENK) and cholecystokinin (CCK) mRNA expressions in the trigeminal ganglia of a rat migraine model and investigates the possible mechanisms by which triptans treat migraine.Methods A total of 24 rats were randomly divided into four groups:normal control group (A),migraine model group(B),rizatriptan control group (C) and rizatriptan treatment group(D).Groups C and D were intragastrically perfused with rizatriptan,1 mg/kg per day.After 7 days,nitroglycerin was subcutaneously injected into the buttocks of the groups B and D to induce migraine.Two hours after nitroglycerin injection,the trigeminal ganglia was isolated.CGRP,PENK and CCK mRNA expressions in the trigeminal ganglia were determined using SYBR Green Ⅰ real-time quantitative PCR.Results The copy number of CGRP mRNA (× 107) in 200 ng total RNA of each group was 0.05 ±0.01,1.30 ±0.52,0.23 ±0.12,0.43 ±0.33 ; The copy number of PENK mRNA (× 103) in 200 ng total RNA of each group was 3.30 ± 1.65,0.34 ±0.14,3.91 ± 2.44,0.71 ± 0.13.The copy number of CGRP mRNA in the trigeminal ganglia of group B was significantly higher than that of group A (q =7.854,P ＜ 0.05) ; CGRP mRNA expressions were significantly lower in the trigeminal ganglia of rats in group D compared with group B (q =5.458,P ＜0.05).Compared with group A,PENK mRNA expressions in the trigeminal ganglia of rats were significantly lower in group B (q =4.478,P ＜ 0.05).PENK mRNA expressions were significantly higher in trigeminal ganglia of rats in group C compared with group D (q =4.838,P ＜ 0.05).CCK mRNA expression in trigeminal ganglia of rats was similar among groups.Conelusions Rizatriptan can decrease the expressions of CGRP in the trigeminal ganglia of the migraine rats and exhibits neurogenic inflammation triggered by CGRP.PENK expressions decrease in the trigeminal ganglia of the migraine rats,weaken the analgesic effects of enkephalin.
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Biochemical and behavioral evidence indicates that the dopaminergic mesolimbic system plays a key role in the mechanisms of reinforcement and reward elicited by alcohol (ethanol) and other drugs of abuse. In addition, the dopaminergic activity of the nigrostriatal pathway has been proposed to determine brain sensitivity to ethanol, a process which could be associated to drug addiction. Besides dopamine, several neurotransmitters and neuromodulators are involved in ethanol reinforcement, including gamma aminobutyric acid (GABA), glutamate, serotonin, acetylcholine and opioid peptides (enkephalins, endorphins and dynorphins). Ethanol and opioids share several pharmacological properties and exhibit similar behavioral effects in animals and humans. These and other studies suggest that the alcohol reinforcing properties are due, at least in part, to the ethanol-induced activation of endogenous opioidergic systems. This activation could in turn increase the hedonic value and the reinforcing effects of the drug. Thus, ethanol-induced changes in opioidergic transmission could contribute to alcohol intoxication and to the neuroadaptive responses produced by the long-lasting exposure to the drug. Opioidergic transmission may be altered by ethanol at different levels, including biosynthesis, release and inactivation of opioid peptides, as well as binding of endogenous opioids to their receptors. Several studies suggest that mu and delta opioid receptors play a key role in ethanol reinforcement and dependence. Therefore, enkephalins and (β-endorphin could mediate ethanol actions in the brain and play a major role in high alcohol drinking behavior. During the last years, our research group has focused on the role of the endogenous opioid systems in these processes. Evidence obtained in our laboratory suggests that enkephalins and (β-endorphin differentially and selectively participate in ethanol reinforcement and dependence.
Evidencias bioquímicas y conductuales indican que el sistema dopaminérgico mesolímbico cumple un papel fundamental en los mecanismos de reforzamiento y recompensa del alcohol (etanol) y otras drogas de abuso. Se ha propuesto también que la actividad de la vía dopaminérgica nigroestriatal determina la sensibilidad cerebral a etanol, lo que parece estar directamente relacionado con los procesos de adicción a la droga. Además de la dopamina, varios neurotransmisores y neuromoduladores están implicados en los mecanismos de reforzamiento del etanol, entre ellos, el ácido gama-aminobutírico (GABA), el glutamato, la serotonina, la acetilcolina y los péptidos opioides (encefalinas, endorfinas y dinorfinas). El alcohol y los opioides comparten características farmacológicas y exhiben efectos similares sobre el comportamiento en animales y en el hombre. Éstos y otros estudios sugieren que las propiedades reforzadoras del etanol se deben, al menos parcialmente, a la activación de los sistemas endógenos de péptidos opioides, proceso que es inducido por el propio alcohol. Esta activación podría, a su vez, aumentar el valor hedónico y los efectos reforzadores de la droga. Los cambios inducidos por etanol sobre la transmisión de opioides podrían contribuir de manera importante a los procesos de intoxicación y a las respuestas neuronales adaptativas que produce el consumo prolongado de la droga. La transmisión opioidérgica puede ser afectada por etanol a distintos niveles, incluyendo la biosíntesis, liberación e inactivación de los opioides endógenos, así como la unión de éstos a sus receptores. Numerosas evidencias sugieren que los receptores opioides mu y delta desempeñan un papel fundamental en el reforzamiento y la dependencia al etanol. Así, las encefalinas y la (β-endorfina actuarían como mediadores fisiológicos de las acciones del etanol en el cerebro, desempeñando un papel crucial en las conductas de alto consumo de la droga. En los últimos años, nuestro grupo se ha centrado en investigar el papel de los sistemas endógenos de péptidos opioides en estos procesos. Las evidencias obtenidas en nuestro laboratorio sugieren que las encefalinas y la (β-endorfina participan en forma diferencial y selectiva en el reforzamiento y la dependencia al etanol.
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ObjectiveTo construct human embryonic kidney cells (HEK293) modified with human preproenkephalin (hPPE) gene.MethodshPPE gene fragments were obtained from recombinant plasmid pcDNA3.1( + )/hPPE by using restriction endonuelease Hind Ⅲ and Not Ⅰ.Homologous recombination of lentivirus and hPPE gene was produced by using recombinant DNA technology.HEK293 cells were then transfected with the recombinant lentivirus vectors.The expression of hPPE gene in HEK293 cells was detected by Western blot.ResultsThe results of DNA sequencing indicated that the positive clone of recombinant lentivirus was completely consistent with sequencing of hPPE in Genebank.The titer of the concentrated virus was 2.07 × 108 TU/ml.GFP fluorescence was not seen in HEK293 cells transfected with the lentiviral vector under fluorescence microscope.A strong fluorescence was seen in HEK293 cells transfected with Ubc-GFP-L.V.empty viral vector.Positive expression of hPPE was demonstrated in HEK293 cells transfected with lentiviral vector by Western blot.Conclusion HEK293 cells modified with hPPE gene were successfully constructed and the target gene hPPE was stably expressed in HEK293 cells.
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Objective To evaluate the feasibility of testing the high methylation of ppENK gene in stool with methylation-specific polymerase chain reaction (MSP) assay in pancreatic cancer diagnosis.Methods Twenty-four fresh stool samples of pancreatic cancer patients and six healthy control samples were collected.The methylation status of ppENK gene in all the stool samples was detected by MSP assay.The positive rate of wild-type ppENK gene in all the stool samples was determined by polymerase chain reaction (PCR).And 10 non wild-type ppENK gene negative pancreatic cancer samples were collected,and K-ras gene mutation was detected by PCR-restriction fragment length polymorphism (RFLP).The single cell suspension of pancreatic cancer PC3 cell line was added into stool sample from the same healthy individual,the positive rate of ppENK gene methylation was detected by MSP assay.The minimum number of pancreatic cancer cell was calculated when methylation was positive.Results The rate of methylation detection in 30 samples was 0 (0/30); and the rate of non-methylation detection was 10％ (3/30).The rate of wild-type ppENK detection was 6.7％ (2/30).By PCR-RFLP assay,eight were successfully amplified and seven had mutation in 12th code of K-ras gene in 10 selected wild-type ppENK gene negative pancreatic cancer samples.The minimum number of pancreatic cancer cells needed for ppENK methylation band positive detected by MSP was 50 cell/ml.Conclusion Detecting ppENK gene methylation status in stool samples of pancreatic cancer patients by MSP assay has not yet become the method of pancreatic cancer screening and diagnosis.
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Objective To investigate the analgesic effect of intrathecal(IT)human bone marrow mesenchymal stem cells(hMSC)genetically modified with human proenkephalin gene(PENK)in a rat model of neuropathic pain.Methods Forty male SD rats weighing 160-180 g in which IT catheters were successfully implanted without complication were randomly divided into 4 gorups(n = 10 each): group A normal control;group B neuropathic pain(NP);group C NP + hMSC-pBABE and group D NP + hMSC-PENK.Neuropathic pain was induced with chronic constrictive injury(CCI).Four loose ligatures were placed on the main stem of sciatic nerve with 4-0 chronic catgut.IT normal saline 10 μl,hMSC-pBABE cell suspension 10 μl(2 × 108-3 × 108/μl)and hMSCPENK cell suspension 10 μl(2 × 108-3 × 108/μl)were injected in group B,C and D respectively on the 3rd day after operation.Paw-withdrawal latency(PWL)to noxious thermal stimulation was measured before(baseline)and at 3,5,7,9 and 14 d after operation.The animals were killed on the 14th day after last PWL measurement.RNA was extracted from the spinal cord for determination of proenkephalin mRNA expression.Results PWL was significantly decreased after operation as compared with the baseline values before operation in group B,C and D.PWL was significantly longer at 7,9,14 d after operation in group D than in group B and C but there was no significant difference in PWL after operation between group B and C.PENK mRNA expression was significantly lower in group B and C than in group A,but was significantly higher in group D than in group B and C.There was no significant difference in PENK mRNA expression between group B and C.Conclusion Intratheccal human bone marrow mesenchymal stem cells genetically modified with human proenkephalin gene can relieve neuropathic pain in rats.
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Objective To construct h n bone marrow mesenchymal stem cell line genetically modified with human proenkephalin gene. Methods The packaging cell line Phoenix-293T was transfected with the recombinant pBABE-PENK vector to aquire virus. The recombinant virus was then collected and used to infect hMSCs. Stable expression of proenkephalin gene and leucine enkephalin protein and the concentration of leucine enkephalin protein were detected by RT-PCR, immunofluorescence and ELISA respectively. Results The expression of proenkephalin gene and leucine enkephalin protein were significantly up-regulated in the hMSC-PENK cells, and the concentration of leucine enkephalin protein was also increased in the culture medium. Conclusion A human mesenchymal stem cell line that expresses proenkephalin gene and secrets enkephalin was successfully established.
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Abstract: Introduction. Epileptic activity modifies the endogenous opioid system by increasing its levels at the end of the ictal phase, and in post-ictal and interictal phases. This increase originates a cortical excitatory effect which suppresses both slow wave sleep and REM. The epileptic activity is initiated with the presence of interictal epileptiform activity, which may be induced through penicillin administration into amygdaline nuclei. Interictal epileptiform activity is a widely employed tool used to determine the localization of epileptic foci characterized by the sudden presence of spikes or acute waves in an electroencephalogram (EEG). In the present work, this tool was used to study the participation of the opioid system in the installation and propagation of epileptic activity induced in temporal lobe amygdala. In the epiloptogenetic study, amygdaline interictal epileptiform activity was used to assess changes induced by opioids and an antagonist in the occurrence of interictal activity using an event histogram. Propagation was studied with the cortical topographic mapping technique, which shows EEG frequency components in a power spectrum, as well as the rhythmic EEG patterns. The aim of the present study was to analyze the effect of enkephalins on epileptiform activity induced with penicillin in tem poral lobe amygdala and its propagation to the cerebral cortex. Method. Fifteen male Wistar rats were submitted to an acute preparation; they were anesthetized with urethane (1.2 g/kg, i.p.). A stainless steel bipolar electrode provided with a cannula was directed toward the left amygdaline basolateral nucleus and a second concentric bipolar electrode to the right amygdaline basolateral nucleus. Two types of cortical recordings were carried out: global mapping and restricted areas. The first consisted of the placement of a 16 stainless steel electrode matrix (in which the electrodes from the vertex were removed) on the scalp, taking care that the tips of the electrodes were in contact with the cortex; this arrangement covered the whole cerebral cortex. The second involved a 4x4mm square matrix consisting of 16 equidistant electrodes placed on the cerebral cortex. The cortical recording was a result of placing this matrix in four different positions so that the whole cerebral cortex was monitored. To monitor cortical recordings, experimental groups were injected penicillin into the amygdaline nuclei. To perform global mapping, enkephalins, [D-ala]-methionine and [D-ala]-leucine, were topically applied into the amygdaline nuclei and naloxone was administered systemically. Analogical signals were recorded in a video-tape and were digitized in parallel with an HP workstation. Off-line analysis was carried as follows: a) information recorded in video-tapes was acquired in a computer designed for this purpose, using amygdaline interictal epileptiform activity to plot event histograms; b) EEG digitized signal, obtained from global mapping, was used to obtain a spectral analysis, consisting of color images maps in time and frequency domains, using RBEAM software. The recording of electrical activity obtained with the square matrix was visually analyzed only. At the end of each experiment, animals were perfused and each brain was fixed intracardially with 10% formaldehyde. To verify the recording and sub-cortical injection sites, the rapid procedure was used. Results. During control stages, cortical records showed slow activity in the form of spindles in all the recording channels; this was due to urethane. Penicillin administration in amygdaline nuclei induced epileptiform activity with a specific pattern: immediate appearance after penicillin application with a gradual increase in amplitude until stabilization was reached within 5-10 minutes of administration. Analyses of global mapping in the frequency domain showed a specific mode of amygdaline interictal epileptiform activity propagation, starting in ipsilateral temporal, prefrontal and fron tal cortices, appearing subsequently in contralateral prefrontal and frontal cortices, and finally in temporal cortex. In the time domain spectrum, an electric dipole generating an interictal spike was found in cerebral cortex. Restricted areas mapping approach showed interictal epileptiform activity and its propagation along the ipsilateral fronto-temporal region. Data revealed an antero-posterior medial cortical activation spreading with decreasing intensity toward occipital regions. Application of enkephalins-[D-ala]-methionine and [D-ala]-leucine produced no epileptic activity, but an increase in basal EEG of cortical epileptiform activity was detected, as well as a decrease in amplitude and frequency of amygdaline epileptiform activity. Naloxone originated a facilitatory effect, since its administration induced focal and generalized electrocorticographic seizures. Conclusions. Focal penicillin is a reliable model of interictal spikes, paroxysms and generalized seizures. The study in rats showed a propagation of epileptic activity to prefrontal cortices prior to contralateral amygdala. Our results showed that enkephalins produced a double effect. First, they originated an increase in basal EEG in temporal cortical areas, as well as a putative participation in propagation mechanisms. Second, they exerted an inhibitory effect on epilepsy installation mechanisms. The inhibitory effect originated by enkephalins was reverted by naloxone.
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Objective To investigate the effects of chronic inflammatory pain induced by injection of complete Freund' s adjuvant (CFA) into the plantar surface of hindpaw on the development of learning and memory and proenkephalin mRNA expression in hippocampus of neonatal rats. Methods sixty neonatal SD rats (6 rats from each of 10 litters) were randomly divided into control and chronic pain group ( n = 30; 3 rats from each of the 10 litters). In chronic pain group CFA 20 ? l was injected subcutaneously into plantar surface of left hindpaw on the 2nd day after birth whereas in control group normal saline 20 ? l was injected instead of CFA. Ten animals (1 rat from each of the 10 litters) in each group were killed on the 10th and 21st day after birth respectively. Hippocampi were removed for determination of proenkephalin mRNA expression by RT-PCR. Ten animals (1 rat from each of the 10 litters) in each group underwent Morris water maze test 3 times a day for 8 days starting from the 21st day after birth. Results The mean latent period before the rats found the hidden platform was significantly longer in chronic pain group than in control group. When the platform was removed the swimming time and distance of the rats in chronic pain group were significantly shorter than those in control group. There was no significant difference in the latent period before the rats found the visible platform between the two groups. The proenkephalin mRNA expression in hippocampus on the 10th and 21st day after birth was significantly lower in chronic pain group than in control group ( P
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Objective To construct the recombinant adeno-associated virus vector with human preproenkephalin gene (rAAV-hPPE). Methods The human preproenkephalin (hPPE) gene was cloned into the adeno-associated virus (AAV) vector plasmid pSNAV which contained neo expression box. The recombinant pSNAV-hPPE was then transfected into BHK cells using lipofectamine?2000. The G418-resistant cells, BHK / SH1, were obtained. The BHK / SH1 cells were infected with HSV1-rc /△UL2 which has the function of packaging the recombinant AAV(rAAV) . After purification, the construction of rAAV-hPPE was achieved.Results The construction of pSNAV-hPPE was confirmed by digestion with restriction enzyme. Southeon bolting was used to detect the virus liters (2.5 ? 1212 v.g /ml) .Conclusion This rAAV-hPPE virus vector with high liter and strong infectivity can be used in transgenic analgesic research.
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Objective To construct an immortalized rat astrocyte strain expressing enkephalin which can be regulated by doxycycline.Methods pRevTet-On and recombinant plasmid pRevTRE/hPPE were transfected into packaging cell PT67 respectively by standard lipofectamine methods.The supernatant containing RevTet-On virus was used to infect immortalized rat astrocyte strain(IAST)and RevTRE/hPPE virus was used to infect the IAST/Tet-On cell.Immortalized rat astrocyte strain that stably expressed Tet-regulated enkephalin was established. hPPE gene expression level of IAST/Tet-On/hPPE cell strain was detected by real time-PCR,immuno- cytochemistry and radio-immunoassay.Results Real time-PCR,immuno-cytochemistry and radio-immunoassay confirmed the level of enkephalin expression was regulated by doxycycline in a dose-dependent manner.Conclusion An immortalized rat astrocyte strain which secrets enkephalin as controlled by doxycycline has been constructed successfully.