Plasma leucine enkephalin content in patients with “Liver-blood deficiency” syndrome and clinical significance

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Distribution，content and action site of leucine－enkephalin in pulmonary artery wall out of lung

When immunohistochemical method was used to investigate the distribution of peptidergic fibers in the pulmonary artery wall of SD rat, L-enkephalin immunoredetive (L-ENK) fibers were easily demonstrated. The contents of L-ENK in pulmonary artery of rat an

Midbrain enkephalin expression in a rat migraine model following intragastric scorpion powder administration

Scorpion has strong analgesic effects, but its analgesic mechanisms remain unclear. This study investigated the effects of scorpion powder on enkephalin expression in the midbrain of rats with nitroglycerin-induced migraine at mRNA and protein levels. Results confirmed that migraine rat abnormal behavior was significantly improved, and proenkephalin mRNA expression was significantly increased following treatment with scorpion. The number of methionine-enkephalin- positive cells in the migraine rats following treatment with scorpion was significantly increased, but no significant difference in the number of leucine-enkephalin-positive cells was detectable compared with migraine and normal rats. Taken together, these results show that scorpion exerts potentially beneficial effects by promoting enkephalin expression in nitroglycerin-induced migraine rats.

Effects of Toutongning capsule on enkephalin expression in a rat migraine headache model

Toutongning capsule is used for the treatment of migraine headaches, and has yielded therapeutically beneficial effects. However, whether Toutongning capsule exerts its effects via endogenous opioid peptides remains poorly understood. This study investigated the effects of Toutongning capsule on enkephalin expression in the midbrain of rats with nitroglycerin-induced migraine headache at the mRNA and protein levels. Results confirmed that proenkephalin mRNA levels were significantly upregulated following treatment with Toutongning capsule. The numbers methionine-enkephalin and leucine-enkephalin-positive cells were significantly greater in the migraine headache rats following treatment with Toutongning capsule compared with the model group. Taken together, these results show that Toutongning capsule exerts potentially beneficial effects by promoting enkephalin expression in nitroglycerin-induced migraine headache rats.

Serotonin样,Substance P样,Enkephalin样反应阳性神经元胞体在大鼠中缝核簇内的分布

本文用PAP法研究了Serotonin样,Substance P样,Leucine—Enkephalin样和Methionine—Enkephalin样反应阳性神经元胞体在大鼠中缝核簇内的分布。证明Serotonin存在于中缝苍白核、中缝隐核、中缝大核、中缝桥核、中央上核和中缝背核等处的某些神经元胞体内。以中、小细胞为主,在中缝大核内有少数大细胞。细胞形态为梭形、圆形、卵圆形以及多极细胞。Substance P样反应阳性胞体在上述中缝核内也被发现,其胞体的大小、形态和分布与serotonin样反应阳性胞体相似,但数目稍少。Leucine—Enkephalin样反应阳性胞体存在于中缝苍白核、中缝隐核、中缝大核、中缝桥核、中缝背核和中央上核内,但Methionine—Enkephalin样反应阳性细胞体仅在中缝苍白核和中缝隐核的吻侧部以及中缝大核内发现。后两者均以大,中型多极细胞为主。

EFFECTS OF ACUPUNCTURE ON THE CONTENTS OF ENKEPHALINS IN DIFFERENT BRAIN REGIONS AND CORTICOSTERONE IN PLASMA OF RATS WITH ENDOTOXIN SHOCK

In this experiment,we have observed the effects of acupuncture on the contents ofenkephalins in different brain regions and corticosterone in plasma of rats with endotoxin shock.Theresults showed that after acupuncture the endogenous enkephalins were significantly decreased(P【0.05)in the midbrain and brain stem of endotoxin shock rats,suggesting the anti-shock of acupuncture might be related to the enkephalins in the midbrain and brain stem.In addition,there was a rising trend in plasma corticosterone of the rats with toxic shock.

Analgesic action of suspended moxibustion in rats with chronic visceral hyperalgesia correlates with enkephalins in the spinal cord

Rats that modeled chronic visceral hyperalgesia received suspended moxibustion at bilateral Tianshu （ST25） and Shangjuxu （ST37） once daily over a period of 7 days. Results show that suspended moxibustion significantly depressed abdominal withdrawal reflex scores and increased enkephalin concentration in the spinal cord. The experimental findings suggest that spinal enkephalins contributed to the analgesic effect of suspended moxibustion in rats with chronic visceral hyperalgesia.

Genome-wide analysis of basic leucine zipper transcription factor families in Arabidopsis thaliana,Oryza sativa and Populus trichocarpa

The basic leucine zipper （bZIP） transcription factors form a large gene family that is important in pathogen defense, light and stress signaling, etc. The Completed whole genome sequences of model plants Arabidopsis （Arabidopsis thaliana）, rice （Oryza sativa） and poplar （Populus trichocarpa） constitute a valuable resource for genome-wide analysis and genomic comparative analysis, as they are representatives of the two major evolutionary lineages within the angiosperms： the monocotyledons and the dicotyledons. In this study, bioinformatics analysis identified 74, 89 and 88 bZIP genes respectively in Arabidopsis, rice and poplar. Moreover, a comprehensive overview of this gene family is presented, including the gene structure, phylogeny, chromosome distribution, conserved motifs. As a result, the plant bZIPs were organized into 10 subfamilies on basis of phylogenetic relationship. Gene duplication events during the family evolution history were also investigated. And it was further concluded that chromosomal/segmental duplication might have played a key role in gene expansion of bZIP gene family.

Effects of electric field stimulation on endogenous enkephalin content in artery wall

After 20min electric field stimulation,L-enkephalin （L-ENK） content in the rabbitear artery declined from the control value of 38.99±17.29 to 12.55±1.96pg/mg of wet tissue（n=12）,and M-enkephalin （M-ENK） content from 134.67±8.11 to 73.68±17.96pg/mg of wet tis-sue （n=6）,both being significantly decreased.This effect was not influenced after blockade ofadrenergic receptor by yohimbine.Contractions of artery strips induced by electric field stimulationwere enhanced distinctly after pretreatment of the artery strips with naloxone,an opioid receptorblocker,and with pronase,but not after pretreatment with trypsin.The results suggested that the re-lease of endogenous enkephalins in the ear artery could be induced by electric field stimulation andx2-receptor might not participate in the release course,The release amount of ENK from the ar-terv strips increased when the stimulus freduency increased within the range of 2 Hz to 8 Hz.

Conceptual Density Functional Theory Study of the Chemical Reactivity Properties and Bioactivity Scores of the Leu-Enkephalin Opioid Peptide Neurotransmitter

The SMD solvation model (Solvation Model based on the Density) and eight density functionals, CAM-B3LYP, LC-ωPBE, M11, MN12SX, N12SX, ωB97, ωB97X, and ωB97XD, were assessed in link with the Def2TZVP basis set for the calculation of the structure of the Leu-Enkephalin Opioid Peptide Neurotransmitter as well as their molecular properties. Through the Conceptual Density Functional Theory (CDFT), the entire chemical descriptors for the system were calculated. The active regions of the molecules necessary for electrophilic, nucleophilic and radical attacks were chosen through linking them with the corresponding Fukui functions. Furthermore, the prediction of the pKa value for the peptide is done with great accuracy as well as the ability of the studied molecule in acting as an efficient inhibitor of the formation of Advanced Glycation Endproducts (AGEs), which comprises of a useful knowledge for the development of drugs for preventing Alzheimer, Diabetes and Parkinson diseases. Lastly, the bioactivity scores for the studied peptides are predicted via various methodologies.

Effects of methionine enkephalin on immune enhancement by reducing myeloid derived suppressor cells and reprogramming liver metabolism in colon cancer mice

OBJECTIVE To investigate enhanced immune function of methionine encephalin(MENK)and its anti-tumor mechanism in CT26 colon cancer mouse model.METHODS 3×10~6CT26 cells were implanted subcutaneously in BALB/c mice.Four days after,MENK was peritoneally administrated at the concentration of 20 mg·kg^(-1) for 14 d.The percentage of MDSCs in bone marrow,spleen,blood,tumor and liver were detected by flow cytometry.Non-esterified fatty acid(NEFA),triglycerides(TG)and total cholesterol(T-CHO)in liver homogenate were tested by a NEFA test kit,a TG test kit and a T-CHO test kit respectively.qRT-PCR and Western blot were used to measure m RNA and protein levels of inflammation-,glycometabolsim-and lipometabolsim-associated indexes in liver.RESULTS MENK decreased percentages of MDSCs in bone marrow,spleen,blood and tumor in colon cancer mice.MENK-treated mice displayed elevated ratio of CD4^+T and CD8^+T cells in spleen as well as increased T and B lymphocytes proliferation.Meanwhile,MENK also ameliorated liver damage reflected by lower levels of GPT and GOT in serum and reduced risks of cancer-associated index including inflammation,high lipid and high glucose.Furthermore,MENK lowered down the levels of NEFA,TG and T-CHO in liver homogenate.MENK treatment decreased expression of p-STAT3,increased expression of p-AKT,IRS1 and Glut4 at protein level as well as reduced lipogenesis-associated genes and elevated glycolysis-associated genes in liver of tumor bearing mice.Also,abated expression of genes associated with MDSCs generation(M-CSF,GM-CSF,IL-6,IL^(-1)β)and migration(S100A9,KC)was observed within shrunken subcutaneous tumor by MENK intervention.CONCLUSION MENK has the ability to strength immune function against colon cancer by reducing MDSCs and improving liver metabolism.

EFFECT OF METHIONINE ENKEPHALIN ON MIGRATION OF MACROPHAGES FROM MICE WITH IMPAIRED LIVER

Objective To observe the effects of methionine enkephalin (M Enk) on migration of macrophages from mice with impaired liver and its immunomodulatory mechanisms. Methods Liver of mice was impaired by feeding CCl 4 and macrophage migration inhibitory factor (MMIF) was produced by Con A stimulated spleen lymphocytes. Inhibition of macrophage migration was measured in reaction system by adding M Enk. Results Migration of macrophages in both liver impaired and control group were suppressed by MMIF, but the suppression might be reversed by adding 1 μmol/L M Enk ( P <0.05).M Enk could significantly inhibit in vitro both of the combination of MMIF with macrophages and production of MMIF from lymphocytes ( P <0.01).Macrophages from liver imparied group showed a higher sensitivity compared to the control group ( P <0.05).Conclusion The study suggests that opioid peptieds play an important role in the modulation of the immune response under stress as liver impairment.

[D-Pen^2,D-Pen^5]enkephalin及Nor-binaltorphimine对吗啡损害小鼠自主选择能力的影响

选择性δ阿片受体激动剂[D-Pen^2,D-Pen^5]enkephalin(DPDPE),k阿片受体拮抗剂nor-bindtorphimine(N-BNI)在测试剂量下对小鼠在丫形迷宫中的自主选择能力无明显影响,但均可显著增强吗啡损害小鼠自主选择能力的作用。DPDPE对吗啡的协同作用还可被δ阿片受体拮抗剂ndltrindole(NTI)所拮抗。

RELATIONSHIP BETWEEN ACUPUNCTURE ANALGESIA AND MET-ENKEPHALIN OR DYNORPHIN

subjective: The effect of 4～5 Hz electroacupuncture (EA) on alterations of both met enkephalin (MEK) and dynorphin (Dyn) in the patient plasma or mouse spinal cord and its relation with analgesic effect were studied.Methods: In acupuncture clinic 10 patients with acute pain were treated with 4 Hz EA at Zusanli(ST 36) and/or Hegu(LI 4) acupoints for 30 min. 20 BALB/C mice were randomly divided into 2 groups: a. EA group(n=10), treated with 4～5 Hz EA at bilateral “Zusanli"(ST 36) for 15 min; b. control group(n=10) treated with no EA, but also restrained for 15 min. Before and after EA or restraining acupoints, the pain threshold of the patients or mice was detected. 10 μl of the patient plasma before and after EA and each mouse spinal cord suspension, of the 2 groups were blotted onto nitrocellulose membrane (NCM) respectively. The protein dot blot signals were detected by immunoreactivity (IR) and using Shimadu TLC Scanner and analyzed statistically. Results: The results showed that an increase in patient plasma MEK IR or Dyn IR and a decrease in mouse spinal MEK IR or Dyn IR could be detected, and the alteration of plasma or spinal MEK IR was more significant than that of plasma or spinal Dyn IR. There was a positive correlation in alteration between plasma or spinal MEK IR and plasma or spinal Dyn IR with respective parallel levels in individuals. The increased plasma MEK IR or the decreased spinal MEK IR was positively or negatively correlated with the analgesic effect, while the correlation between plasma or spinal Dyn IR and analgesic effect was insignificant. Conclusion: The results suggest that under lower frequency EA the met enkephalin may play an important role in analgesia.

Effect of Intracerebral Injection of Substance P on Pain Threshold,Electroacupuncture Analgesia and Met-enkephalin Level in Rat Brain

Radioimmunoassay was employed to dctermine the immunorcactivc met-enkephalin(ir-MEK)contents of several brain regions .The pain threshold(PT)andir-MEK content of striatum and hypothalamus increased markedly afterintraoerebroventricular injection of substance P(SP)in the dosage of lμgg/10μl ,but nosignificant change of ir-MEK content in hippocampus was observed.When theperiaqueductal gray(PAG)was destroyed with electrolysis,the increase of PT andir-MEK content in the two brain regions after SP injection was obviouslysuppressed(P【0.05-0.01).The PT and ir-MEK content of hypothalamus andstriatum would also increase after intra-PAG injection of SP(0.4μg/4μl),electroacupuncture or intraperitoneal injection of morphine.The ir-MEK content ofhippocampus could also increase after intra-PAG injection of SP or intraperitoncal injec-tion of morphine.The combination of intra-PAG injection of SP andelectroacupuncture caused a more marked elevation of PT and ir-MEK content of the 3brain regions than either of the two used singly could(P【0.01 ).The combined effect ofintra-PAG injection of SP and intraperitoneal injection of morphine was similar to thatof either of the two used singly(P】0.05).These facts indicate that thc analgesic effect ofSP activated by PAG was mediated through the increase of cerebral met-cnkcphalin con-tent,and there was a synergic effect between SP and elcctroacupuncturc.

Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway

Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine＇s action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin （mTOR） complex-1 （mTORC1）. Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine （400 μmol·kg^-1·h^-1） for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 （S6K1） and eukaryotic initiation factor （elF） 4E-binding protein-1 （4E-BP1） phosphorylation in gastrocnemius and masseter muscles （P 〈 0.05）, but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTQRC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of elF2α and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation.

Dietary Leucine Requirement for Juvenile Large Yellow Croaker Pseudosciaena crocea (Richardson,1846)

Dietary leucine requirement for juvenile large yellow croaker, Pseudosciaena crocea Richardson 1846 (initial body weight 6.0 g±0.1 g) was determined using dose-response method.Six isonitogenous (crude protein 43%) and isoenergetic (19 kJ g-1) practical diets containing six levels of leucine (Diets 1-6) ranging from 1.23% to 4.80% (dry matter) were made at about 0.7% increment of leucine.Equal amino acid nitrogen was maintained by replacing leucine with glutamic acid.Triplicate groups of 60 individuals were fed to apparent satiation by hand twice daily (05:00 and 17:30).The water temperature was 26-32℃, salinity 26-30 and dissolved oxygen approximately 7 mg L-1 during the experimental period.Final weight (FW) of large yellow croaker initially increased with increasing level of dietary leucine but then decreased at further higher level of leucine.The highest FW was obtained in fish fed diet with 3.30% Leucine (Diet 4).FW of fish fed the diet with 4.80% Leucine (Diet 6) was significantly lower than those fed Diet 4.However, no significant differences were observed between the other dietary treatments.Feed efficiency (FE) and whole body composition were independent of dietary leucine contents (P>0.05).The results indicated that leucine was essential for growth of juvenile large yellow croaker.On the basis of FW, the optimum dietary leucine requirement for juvenile large yellow croaker was estimated to be 2.92% of dry matter (6.79% of dietary protein).

Dietary Leucine Requirement of Juvenile Japanese Seabass(Lateolabrax Japonicus)

A 56-day feeding trial was conducted to examine the dietary leucine requirement of juvenile Japanese seabass in sea- water floating net cages （1.5 m × 1.5 m × 2.0 m）. Six isonitrogenous （crude protein 40%） and isoenergetic （gross energy 20 kJ g-1） diets were formulated to contain different concentrations of leucine （0.9%, 1.49%, 2.07%, 2.70%, 3.30% and 3.88% of dry matter）. Crys- talline L-amino acids were supplemented to simulate the whole body amino acid pattern of Japanese seabass except for leucine. Three groups （30 fish individuals each, 8.0g±0.20g in initial weight） were fed to apparent satiation at 5：00 and 17：30 every day. During the experimental period, the water temperature ranged from 26 to 32℃ and salinity from 26 to 30, and the dissolved oxygen was maintained at 7mgL-l. The results showed that weight gain （WG）, nitrogen retention （NR）, feed efficiency （FE） and protein efficiency ratio （PER） were significantly increased when dietary leucine was increased from 0.90% to 2.70% of dry matter, and then declined. WG was the highest when fish were fed D4 containing 2.70% of leucine. No significant differences were observed in body composition among dietary treatments （P 〉 0.05）. Considering the change of WG, the optimum dietary leucine requirement of juve- nile Japanese seabass was either 2.39% of dry matter or 5.68% of dietary protein.

Application of quartz crystal nanobalance in conjunction with a net analyte signal based method for simultaneous determination of leucine, isoleucine and valine

The aim of the present investigation was to develop a biosensor for the detection of amino acids, Leucine, Isoleucine and Valine based on a quartz crystal nanobalance. leucine (Leu), isoleucine (Ile), and valine (Val) were selectively determined by quartz crystal nanobalance (QCN) sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including Leu, Ile and Val compounds. The selection of the optimal time range involved the calculation of the net analyte sig-nal regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of PRESS value was carried out by applying a moving window strategy. On the base of obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. The results showed that the method was successfully applied for the determina-tion of Leu, Ile and Val.

NMR Analysis of the Structure of Leucine Enkephalin in Solution

To investigate the interaction between membranes and polypeptides, the structure of the polypeptide must be known first. Enkephalin, a natural ligand for opiate receptors is composed of the pentapeptides H 2N Tyr Gly Gly Phe Met OH and H 2N Tyr Gly Gly Phe Leu OH. Two dimensional 1 H NMR techniques can be used to determine the structure of Leucine Enkephalin in solution and to build a foundation for research on the interaction between Enkephalin and the membrane. Complete proton resonance assignments were obtained using 2 D COSY, TOCSY, and NOESY techniques.