Novel Cyclic Endomorphin Analogues with Multiple Modifications and Oligoarginine Vector Exhibit Potent Antinociception with Reduced Opioid-like Side Effects.

Endomorphins (EMs) are potent pharmaceuticals for the treatment of pain. Herein, we investigated several novel EM analogues with multiple modifications and oligoarginine conjugation. Our results showed that analogues 1-6 behaved as potent µ-opioid agonists and enhanced stability and lipophilicity. Analogues 5 and 6 administered centrally and peripherally induced significant and prolonged antinociceptive effects in acute pain. Both analogues also produced long-acting antiallodynic effects against neuropathic and inflammatory pain. Furthermore, they showed a reduced acute antinociceptive tolerance. Analogue 6 decreased the extent of chronic antinociceptive tolerance, and analogue 5 exhibited no tolerance at the supraspinal level. Particularly, they displayed nontolerance-forming antinociception at the peripheral level. In addition, analogues 5 and 6 exhibited reduced or no opioid-like side effects on gastrointestinal transit, conditioned place preference (CPP), and motor impairment. The present investigation established that multiple modifications and oligoarginine-vector conjugation of EMs would be helpful in developing novel analgesics with fewer side effects.

Enhancement of Anti-Tumoral Immunity by ß-Casomorphin-7 Inhibits Cancer Development and Metastasis of Colorectal Cancer.

ß-Casomorphin-7 (BCM) is a degradation product of ß-casein, a milk component, and has been suggested to affect the immune system. However, its effect on mucosal immunity, especially anti-tumor immunity, in cancer-bearing individuals is not clear. We investigated the effects of BCM on lymphocytes using an in vitro system comprising mouse splenocytes, a mouse colorectal carcinogenesis model, and a mouse orthotopic colorectal cancer model. Treatment of mouse splenocytes with BCM in vitro reduced numbers of cluster of differentiation (CD) 20+ B cells, CD4+ T cells, and regulatory T cells (Tregs), and increased CD8+ T cells. Administration of BCM and the CD10 inhibitor thiorphan (TOP) to mice resulted in similar alterations in the lymphocyte subsets in the spleen and intestinal mucosa. BCM was degraded in a concentration- and time-dependent manner by the neutral endopeptidase CD10, and the formed BCM degradation product did not affect the lymphocyte counts. Furthermore, degradation was completely suppressed by TOP. In the azoxymethane mouse colorectal carcinogenesis model, the incidence of aberrant crypt foci, adenoma, and adenocarcinoma was reduced by co-treatment with BCM and TOP. Furthermore, when CT26 mouse colon cancer cells were inoculated into the cecum of syngeneic BALB/c mice and concurrently treated with BCM and TOP, infiltration of CD8+ T cells was promoted, and tumor growth and liver metastasis were suppressed. These results suggest that by suppressing the BCM degradation system, the anti-tumor effect of BCM is enhanced and it can suppress the development and progression of colorectal cancer.

Keratin Scaffolds Containing Casomorphin Stimulate Macrophage Infiltration and Accelerate Full-Thickness Cutaneous Wound Healing in Diabetic Mice.

Impaired wound healing is a major medical challenge, especially in diabetics. Over the centuries, the main goal of tissue engineering and regenerative medicine has been to invent biomaterials that accelerate the wound healing process. In this context, keratin-derived biomaterial is a promising candidate due to its biocompatibility and biodegradability. In this study, we evaluated an insoluble fraction of keratin containing casomorphin as a wound dressing in a full-thickness surgical skin wound model in mice (n = 20) with iatrogenically induced diabetes. Casomorphin, an opioid peptide with analgesic properties, was incorporated into keratin and shown to be slowly released from the dressing. An in vitro study showed that keratin-casomorphin dressing is biocompatible, non-toxic, and supports cell growth. In vivo experiments demonstrated that keratin-casomorphin dressing significantly (p < 0.05) accelerates the whole process of skin wound healing to the its final stage. Wounds covered with keratin-casomorphin dressing underwent reepithelization faster, ending up with a thicker epidermis than control wounds, as confirmed by histopathological and immunohistochemical examinations. This investigated dressing stimulated macrophages infiltration, which favors tissue remodeling and regeneration, unlike in the control wounds in which neutrophils predominated. Additionally, in dressed wounds, the number of microhemorrhages was significantly decreased (p < 0.05) as compared with control wounds. The dressing was naturally incorporated into regenerating tissue during the wound healing process. Applied keratin dressing favored reconstruction of more regular skin structure and assured better cosmetic outcome in terms of scar formation and appearance. Our results have shown that insoluble keratin wound dressing containing casomorphin supports skin wound healing in diabetic mice.

MCRT, a multifunctional ligand of opioid and neuropeptide FF receptors, attenuates neuropathic pain in spared nerve injury model.

Chimeric peptide MCRT (YPFPFRTic-NH2 ) was a multifunctional ligand of opioid and neuropeptide FF (NPFF) receptors and reported to be potentially antalgic in acute tail-flick test. Here, we developed spared nerve injury (SNI) model to explore its efficacy in chronic neuropathic pain. Analgesic tolerance, opioid-induced hyperalgesia and gastrointestinal transit were measured for safety evaluation. Intracerebroventricular (i.c.v.) and intraplantar (i.pl.) injections were conducted as central and peripheral routes, respectively. Results demonstrated that MCRT alleviated neuropathic pain effectively and efficiently, with the ED50 values of 4.93 nmol/kg at the central level and 3.11 nmol/kg at the peripheral level. The antagonist blocking study verified the involvement of mu-, delta-opioid and NPFF receptors in MCRT produced anti-allodynia. Moreover, the separation of analgesia from unwanted effects was preliminarily achieved and that MCRT caused neither analgesic tolerance nor hyperalgesia after chronic i.c.v. administration, nor constipation after i.pl. administration. Notably, the local efficacy of MCRT in SNI mice was about one thousandfold higher than morphine and ten thousandfold higher than pregabalin, indicating a great promise in the future treatment of neuropathic pain.

Insulin Bidirectionally Alters NAc Glutamatergic Transmission: Interactions between Insulin Receptor Activation, Endogenous Opioids, and Glutamate Release.

Human fMRI studies show that insulin influences brain activity in regions that mediate reward and motivation, including the nucleus accumbens (NAc). Insulin receptors are expressed by NAc medium spiny neurons (MSNs), and studies of cultured cortical and hippocampal neurons suggest that insulin influences excitatory transmission via presynaptic and postsynaptic mechanisms. However, nothing is known about how insulin influences excitatory transmission in the NAc. Furthermore, insulin dysregulation accompanying obesity is linked to cognitive decline, depression, anxiety, and altered motivation that rely on NAc excitatory transmission. Using whole-cell patch-clamp and biochemical approaches, we determined how insulin affects NAc glutamatergic transmission in nonobese and obese male rats and the underlying mechanisms. We find that there are concentration-dependent, bidirectional effects of insulin on excitatory transmission, with insulin receptor activation increasing and IGF receptor activation decreasing NAc excitatory transmission. Increases in excitatory transmission were mediated by activation of postsynaptic insulin receptors located on MSNs. However, this effect was due to an increase in presynaptic glutamate release. This suggested feedback from MSNs to presynaptic terminals. In additional experiments, we found that insulin-induced increases in presynaptic glutamate release are mediated by opioid receptor-dependent disinhibition. Furthermore, obesity resulted in a loss of insulin receptor-mediated increases in excitatory transmission and a reduction in NAc insulin receptor surface expression, while preserving reductions in transmission mediated by IGF receptors. These results provide the first insights into how insulin influences excitatory transmission in the adult brain, and evidence for a previously unidentified form of opioid receptor-dependent disinhibition of NAc glutamatergic transmission.SIGNIFICANCE STATEMENT Data here provide the first insights into how insulin influences excitatory transmission in the adult brain, and identify previously unknown interactions between insulin receptor activation, opioids, and glutamatergic transmission. These data contribute to our fundamental understanding of insulin's influence on brain motivational systems and have implications for the use of insulin as a cognitive enhancer and for targeting of insulin receptors and IGF receptors to alter motivation.

ß-Casomorphin: A complete health perspective.

ß-Casomorphin-7 (BCM-7) is a heptapeptide dietary molecule derived from the digestion of the ß-casein of dairy and dairy products. In this review, we have covered the extensive details about BCM and its derived peptides out of the gastrointestinal and enzymatic digestion of milk and milk products, its structure and properties, and its immunological aspects related to human health among infants and adults of both genders. We have left judgment about BCM's pros and cons to the reader by describing the details in a cyclopedic perspective. In addition, a section on the possible ways to detect BCMs from their sources using proteomics, genome-based techniques, such as PCR and aptamers, and other analytical techniques equip the reader to get an idea about the details of the diagnostics available and possible applications in future. Overall, this review will provide information to the end-users of milk and milk products to enable them to make their own decisions about BCMs.

Cyclic derivative of morphiceptin Dmt-cyclo-(D-Lys-Phe-D-Pro-Asp)-NH2(P-317), a mixed agonist of MOP and KOP opioid receptors, exerts anti-inflammatory and anti-tumor activity in colitis and colitis-associated colorectal cancer in mice.

Endogenous opioid system is involved in the maintenance of the intestinal homeostasis. Recently, we proved that stimulation of opioid receptors using P-317, a cyclic morphiceptin analog, resulted in the alleviation of acute colitis in mice. The aim of the current study was to assess the effect of P-317 during colitis and colitis-associated colorectal cancer in mice. Colitis was induced by addition of dextran sodium sulfate (DSS) into drinking water. Colitis-associated colorectal cancer was induced by a single intraperitoneal injection of azoxymethane (AOM) and subsequent addition of DSS into drinking water (week 2, 5, 8). During macroscopic damage evaluation the samples were collected and used for biochemical (MPO activity assay), molecular (qPCR and western blot) and histological studies. In experimental colitis, P-317 induced an anti-inflammatory response as indicated by macroscopic and microscopic scores. In the colitis-associated colorectal cancer model, a significant difference in colorectal tumor development was observed between vehicle- and P-317-treated mice. P-317 decreased the total number of colonic tumors and inhibited MPO activity. Hematoxylin and eosin staining confirmed anti-tumor activity of P-317. The expression of TNF-α was decreased in P-317-treated mice as compared to the vehicle-treated group. P-317 decreased proliferation as well as ß-catenin expression in tumors. P-317, a mixed MOP and KOP receptor agonist, induced an anti-inflammatory response in experimental colitis and decreased tumor development in colitis-associated colorectal cancer in mice.

The protective effects of human milk-derived peptides on the pancreatic islet biology.

Several epidemiological studies support the protective role of breastfeeding in reducing the risk for type 1 diabetes. Human breast milk is the perfect nutrition for infants and contains many complex proteins, lipids and carbohydrates. In this study, we examined the physiological effects of human milk-derived opioid peptides, ß-casomorphins (BCM), and compared them with bovine-milk-derived opioid peptides on pancreatic hormone regulation and ß-cell regeneration. Exposure of wild-type zebrafish embryos to 50 µg/ml of human BCM-5 and -7 from 3 days post fertilisation until 6 days post fertilisation resulted in an increased insulin domain of expression while exposure to bovine BCM-5 and -7 significantly reduced the insulin domain of expression as analysed by whole-mount in situ hybridisation. These changes may be accounted for by reduced insulin expression or ß-cell number and were mitigated by the µ-opioid receptor antagonist, naloxone. The effect of BCM on ß-cell regeneration was assessed following ablation of ß-cells in Tg (ins: CFP-NTR) zebrafish from 3 days post fertilisation to 4 days post fertilisation, followed by exposure of bovine and human BCM-5 and -7 (50 µg/ml) from 4 days post fertilisation until 7 days post fertilisation. The regenerative capacity of ß-cells was not impeded following exposure to human BCM-5 and -7, whereas the capacity of ß-cells to regenerate following bovine BCM-5 and -7 exposure was reduced. Our data suggest that human BCM-5 and -7 may promote ß-cell development and enable the regeneration of ß-cells, while the bovine-milk-derived peptides, BCM-5 and -7, play an opposite role. These data may provide some biological explanation for the protective effect of breastfeeding on the development of type 1 diabetes.

Effect of ß-casomorphin-7 on myocardial hypertrophy in hyperthyroidism-induced cardiomyopathy.

OBJECTIVE: The purpose of this study was to investigate the effect of ß-casomorphin-7 (ß-CM-7) on myocardial hypertrophy (MH) in hyperthyroidism-induced cardiomyopathy in vivo and in vitro. MATERIALS AND METHODS: Thirty C56BL/6 mice were randomly divided into three groups: control group, hyperthyroidism group, and ß-CM-7 treatment group. An animal model of cardiac hypertrophy of hyperthyroid heart disease (HHD) was constructed by continuous intraperitoneal injection of 100 µg of L-thyroxine (L-Thy) for 28 days, and the serum TT3 and TT4 concentrations were measured. After that, myocardial specimens were collected to measure left and right ventricular MH index, and the myocardial cell structure was observed under hematoxylin and eosin (HE) staining. Thereafter, Masson staining was adopted to determine collagen volume fraction, and hydroxylamine method was used to measure superoxide dismutase (SOD) activity, Meanwhile, DTNB direct method was applied to measure GSH-Px activity, thio-malonylurea method was utilized to measure malondialdehyde (MDA) content, and the level of reactive oxygen species (ROS) was detected by flow cytometry. Finally, the expressions of oxidative stress (OS) and inflammation-related factors in vivo and the nuclear factor-κB (NF-κB) pathway in vitro were detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Compared with those in control group, TT3 and TT4 were remarkably increased, the structure of myocardial cells was disordered, the interstitial fibrosis and the ventricular MH index were significantly increased, the OS and inflammatory responses were increased, and the NF-κB pathway was activated in the Hyperthyroidism group. In the ß-CM-7 group, the content of TT3 and TT4 was decreased, the myocardial cell structure was slightly disturbed, the fibrosis and the ventricular MH index were reduced, OS and inflammatory response were reduced, and the NF-κB pathway was inhibited. CONCLUSIONS: ß-CM-7 can prevent and treat MH in mice with L-Thy-induced HHD probably through regulating the NF-κB signaling pathway.

Antifungal and anti-biofilm activity of designed derivatives from kyotorphin.

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins-KTP-NH2, KTP-NH2-DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH2, N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg-were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates-Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S-obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH2, in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application.

Cyclic derivatives of morphiceptin possess anti-transit effect in the gastrointestinal tract and alleviate abdominal pain in mice.

BACKGROUND: Irritable bowel syndrome (IBS) is a chronic condition with recurring gastrointestinal (GI) symptoms: altered motility and abdominal pain. As endogenous opioid system participates in pain perception and in the control of GI peristalsis, opioids have been proposed as a promising therapy in IBS. In a previous study, we observed that morphiceptin derivative, P-317 (Dmt-cyclo-(D-Lys-Phe-D-Pro-Asp)-NH2), presents promising features to be applied in IBS. In this project, we tested whether modifications in cyclic morphiceptin-based structure: fluorination (compound 1) or peptide bond reduction (compound 2) improve pharmacological effect. METHODS: We evaluated tested derivatives in the mouse GI system under physiological (GI transit) and pathophysiological (castor oil diarrhea, stress-induced hypermotility, visceral pain) conditions. RESULTS: Both compounds prolonged GI transit. Compound 1 and P-317 inhibited upper GI transit and motility of the colon; compound 2 remained inactive. Compound 1 and P-317 inhibited hypermotility in stressed mice and delayed the acute diarrhea in comparison to control. Only P-317 exerted antinociceptive effect. None of tested derivatives, similar to P-317, affected locomotor activity. CONCLUSIONS: Compound 1 is equally effective as P-317 in the mouse GI tract. The peptide bond reduction decreased the activity of compound 2. Fluorination appears to be an efficient way to increase the effects of morphiceptin analogs in the GI tract.

The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor.

BACKGROUND: Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles. METHODS: Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. RESULTS: We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. CONCLUSION: We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.

ß-Casomorphin-7 Ameliorates Sepsis-Induced Acute Kidney Injury by Targeting NF-κB Pathway.

BACKGROUND The aim of this study was to investigate the protective effect of ß-casomorphin-7 (ß-CM-7) and its possible mechanisms on acute kidney injury (AKI). MATERIAL AND METHODS Rats were randomly divided into a sham group, a cecal ligation and puncture (CLP) group, and a CLP+ß-CM-7 group. Kidney index, kidney function, and histopathology changes were assessed. The expression of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (Kim-1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and p-IκBα in kidney tissues were detected by Western blotting. Inflammatory and oxidative stress factors were detected by ELISA kits. RESULTS The results showed that treatment with ß-CM-7 reduced the levels of creatinine (Cre), blood urea nitrogen (BUN), NGAL, and Kim-1 induced by CLP, weakening the pathological damage. In the CLP + ß-CM-7 group, the tumor necrosis factor-α (TNF-α) level and the DNA-binding activity of NF-κB p65 were significantly reduced and the interleukin-10 (IL-10) level was significantly increased compared with the CLP group. ß-CM-7 decreased the expression of p-IκBα/IκBα. In addition, ß-CM-7 increased the activity of superoxide dismutase (SOD) and decreased the level of malondialdehyde (MDA) in kidney tissue. CONCLUSIONS ß-CM-7 attenuated sepsis-induced AKI through reducing inflammation and oxidative stress and by inhibition of nuclear factor (NF)­κB activities. This study provides a new therapeutic agent for attenuating sepsis-induced kidney injury.

Role of Milk-Derived Opioid Peptides and Proline Dipeptidyl Peptidase-4 in Autism Spectrum Disorders.

Opioid peptides released during digestion of dietary proteins such as casein, were suggested to contribute to autism development, leading to the announcement of opioid excess hypothesis of autism. This paper examines role of enzyme proline dipeptidyl peptidase-4 (DPPIV; EC 3.4.14.5) and it is exogenous substrate, ß-casomorphin-7 (BCM7) in autism etiology. Our study included measurements of DPPIV and BCM7 concentrations in serum and urine, which were analyzed with ELISA assays and activity of DPPIV was measured by colorimetric test. The effect of opioid peptides from hydrolysed bovine milk on DPPIV gene expression in peripheral blood mononuclear cells (PBMC) in autistic and healthy children was determined using the Real-Time PCR (Polymerase Chain Reaction) method. Our research included 51 healthy children and 86 children diagnosed with autism spectrum disorder (ASD, ICDF84). We determined that the concentration of BCM7 in serum was significantly, 1.6-fold, higher in the ASD group than in controls (p < 0.0001). Concentration of DPPIV was found to also be significantly higher in serum from ASD children compared to the control group (p < 0.01), while we did not notice significant difference in enzymatic activity of serum DPPIV between the two study groups. We confirmed correlation according to the gender between analyzed parameters. The inspiration for this study emanated from clinical experience of the daily diet role in relieving the symptoms of autism. Despite this, we have concluded that milk-derived opioid peptides and DPPIV are potentially factors in determining the pathogenesis of autism; conducted studies are still limited and require further research.

ß-Casomorphin increases fat deposition in broiler chickens by modulating expression of lipid metabolism genes.

ß-Casomorphin is an opioid-like bioactive peptide derived from ß-casein of milk that plays a crucial role in modulating animal's feed intake, growth, nutrient utilization and immunity. However, the effect of ß-casomorphin on lipid metabolism in chickens and its mechanism remain unclear. The aim of this study was to investigate the effects of ß-casomorphin on fat deposition in broiler chickens and explore its mechanism of action. A total of 120 21-day-old Arbor Acres male broilers (747.94±8.85 g) was chosen and randomly divided into four groups with six replicates of five birds per replicate. Three groups of broilers were injected with 0.1, 0.5 or 1.0 mg/kg BW of ß-casomorphin in 1 ml saline for 7 days, whereas the control group received 1 ml saline only. The results showed that subcutaneous administration of ß-casomorphin to broiler chickens increased average daily gain, average daily feed intake and fat deposition, and decreased feed : gain ratio (P<0.05). The activity of malate dehydrogenase in the pectoral muscle, liver and abdominal adipose tissue was also increased along with the concentrations of insulin, very-low-density lipoprotein and triglyceride in the plasma (P<0.05). The activity of hormone-sensitive lipase in the liver and abdominal adipose tissue and the concentration of glucagon in the plasma were decreased by injection with ß-casomorphin (P<0.05). Affymetrix gene chip analysis revealed that administering 1.0 mg/kg BW ß-casomorphin caused differential expression of 168 genes in the liver with a minimum of fourfold difference. Of those, 37 genes are directly involved in lipid metabolism with 18 up-regulated genes such as very low density lipoprotein receptor gene and fatty acid synthase gene, and 19 down-regulated genes such as lipoprotein lipase gene and low density lipoprotein receptor gene. In conclusion, ß-casomorphin increased growth performance and fat deposition of broilers. Regulation of fat deposition by ß-casomorphin appears to take place through changes in hormone secretion and enzyme activities by controlling the gene expression of lipid metabolism and feed intake, increasing fat synthesis and deposition.

In vivo endogenous proteolysis yielding beta-casein derived bioactive beta-casomorphin peptides in human breast milk for infant nutrition.

Objective Beta-casein is a major protein in breast milk and an important source for several bioactive peptides that are encrypted within the sequence. Beta-casomorphins (BCMs) are short-chain proteolytic peptides that are derived from the beta-casein protein and have opioid effects in newborns. Human milk is known to contain naturally occurring milk-protein-derived bioactive peptides but the identification of naturally occurring beta-casein-derived BCMs in human breast milk has been limited due to difficulties in the detection of BCM peptides, which are small and circulate in low concentrations. Methods The present study aimed to identify the naturally occurring BCM peptides from beta-casein in human breast milk using liquid chromatography-tandem mass spectrometry. The BCM peptides identified in the breast milk were analysed to predict the milk proteases responsible for the cleavage patterns using a computational tool EnzymePredictor. Results In-depth peptidomics analysis of breast milk samples that were collected at different lactation stages during human lactation revealed the presence of BCMs including BCM-8, -9, -10, and -11 as well as precursors and truncated forms of the original peptide, which suggests that milk protease activity in the mammary gland generates biologically relevant BCMs. Conclusions To our knowledge, this is the first report to describe the presence of naturally occurring human BCM-10 and -11 in breast milk. Our study provides evidence of beta-casein-derived BCM peptides in human milk before infant digestion. Proteases that are present in milk are likely specific in their proteolysis of beta-casein. The identified bioactive BCM-8, -9, -10, and -11 as well as the precursor peptides meet the structural requirements to elicit opioid, immunomodulatory, antioxidative, and satiety functions in newborns.

The protective effect of beta-casomorphin-7 via promoting Foxo1 activity and nuclear translocation in human lens epithelial cells.

PURPOSE: To investigate the protective effect of beta-casomorphin-7 (ß-CM-7) in oxidative stressed human lens epithelial cells (HLECs) and to explore the possible mechanism for oxidative stress in HLECs induced by high glucose. METHODS: We used HLECs to determine the effect of different concentrations of ß-CM-7 on cell viability by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolimol/L bromide (MTT) assay. We used flow cytometry to determine the content of reactive oxygen species (ROS) induced by oxidative stress and a bioassay kit to determine the oxidant malondialdehyde (MDA) and antioxidant enzyme superoxide dismutase (SOD) levels. We used Western blotting and an immunofluorescence assay to determine the expression of Forkhead box o1 (Foxo1), SP1, and the related protein glutathione peroxidase (GSH-px) at the molecular biology level as well as their intracellular localization. RESULTS: The expression of Foxo1 and SP1 was weakly expressed when the glucose concentration was 40 mM/L, but was highly expressed when cells were pre-treated with an appropriate concentration of ß-CM-7. After pre-treatment with ß-CM-7, the cells treated with 40 mM/L glucose for 48 h showed Foxo1 was transferred to the nucleus, and the expression of SP1 was increased. The content of ROS and MDA in the HLECs that were pre-treated with ß-CM-7 was lower than in those that was not pre-treated (p <0.05). Accordingly, SOD was elevated in the cells pre-treated with ß-CM-7. The relative expression of GSH-px increased with increases of Foxo1 and SP1. CONCLUSION: ß-CM-7 protects HLECs from oxidative damage by upregulating the relative expression of Foxo1 and promoting Foxo1 nuclear translocation.

Supraspinal inhibitory effects of chimeric peptide MCRT on gastrointestinal motility in mice.

OBJECTIVES: Chimeric peptide MCRT, based on morphiceptin and PFRTic-NH2 , was a bifunctional ligand of µ- and δ-opioid receptors (MOR-DOR) and produced potent analgesia in tail-withdrawal test. The study focused on the supraspinal effects of morphiceptin, PFRTic-NH2 and MCRT on gastrointestinal motility. Moreover, opioid receptor antagonists, naloxone (non-selective), cyprodime (MOR selective) and naltrindole (DOR selective) were utilized to explore the mechanisms. METHODS: Intracerebroventricular administration was achieved via the implanted cannula. Gastric emptying and intestinal transit were measured to evaluate gastrointestinal motility. KEY FINDINGS: (1) At supraspinal level, morphiceptin, PFRTic-NH2 and MCRT significantly decreased gastric emptying and intestinal transit; (2) MCRT at 1 nmol/mouse, far higher than its analgesic dose (ED50  = 29.8 pmol/mouse), failed to regulate the gastrointestinal motility; (3) MCRT-induced gastrointestinal dysfunction could be completely blocked by naloxone and naltrindole, but not affected by cyprodime. CONCLUSIONS: (1) Morphiceptin and PFRTic-NH2 played important roles in the regulation of gastrointestinal motility; (2) MCRT possessed higher bioactivity of pain relief than gastrointestinal regulation, suggesting its promising analgesic property; (3) MCRT-induced motility disorders were sensitive to DOR but not to MOR blockade, indicating the pain-relieving specificity of speculated MOR subtype or splice variant or MOR-DOR heterodimer.

Identification of the First Marine-Derived Opioid Receptor "Balanced" Agonist with a Signaling Profile That Resembles the Endorphins.

Opioid therapeutics are excellent analgesics, whose utility is compromised by dependence. Morphine (1) and its clinically relevant derivatives such as OxyContin (2), Vicodin (3), and Dilaudid (4) are "biased" agonists at the µ opioid receptor (OR), wherein they engage G protein signaling but poorly engage ß-arrestin and the endocytic machinery. In contrast, endorphins, the endogenous peptide agonists for ORs, are potent analgesics, show reduced liability for tolerance and dependence, and engage both G protein and ß-arrestin pathways as "balanced" agonists. We set out to determine if marine-derived alkaloids could serve as novel OR agonist chemotypes with a signaling profile distinct from morphine and more similar to the endorphins. Screening of 96 sponge-derived extracts followed by LC-MS-based purification to pinpoint the active compounds and subsequent evaluation of a mini library of related alkaloids identified two structural classes that modulate the ORs. These included the following: aaptamine (10), 9-demethyl aaptamine (11), demethyl (oxy)-aaptamine (12) with activity at the δ-OR (EC50: 5.1, 4.1, 2.3 µM, respectively) and fascaplysin (17), and 10-bromo fascaplysin (18) with activity at the µ-OR (EC50: 6.3, 4.2 µM respectively). An in vivo evaluation of 10 using δ-KO mice indicated its previously reported antidepressant-like effects are dependent on the δ-OR. Importantly, 17 functioned as a balanced agonist promoting both G protein signaling and ß-arrestin recruitment along with receptor endocytosis similar to the endorphins. Collectively these results demonstrate the burgeoning potential for marine natural products to serve as novel lead compounds for therapeutic targets in neuroscience research.

MCRT, a chimeric peptide based on morphiceptin and PFRTic-NH2, regulates the depressor effects induced by endokinin A/B.

The interactions of the chimeric peptide MCRT (YPFPFRTic-NH2), based on morphiceptin and neuropeptide FF derivative PFRTic-NH2, on the effects of endokinin A/B (EKA/B) on mean arterial blood pressure of the urethane-anaesthetized rat have been investigated in the absence and presence of tachykinin receptor antagonists, naloxone and NO synthase inhibitors. While MCRT produced dose dependent decreases in mean arterial pressure, in its presence only a small but statistically insignificant decreases in the magnitude and the time course of the depressor effect of EKA/B (10nmol/kg) were observed. MCRT had little influence on the depressor effect of EKA/B (1 nmol/kg), but strongly potentiated that of EKA/B (100nmol/kg). The tachykinin NK1 receptor antagonist SR140333B (1mg/kg) and the NK3 antagonist SR142891 (2.79mg/kg) both reduced the hypotensive effects of EKA/B and MCRT alone and blocked those of the two peptides in combination. The NK2 antagonist GR159897 (4mg/kg) partially blocked the depressor effects of EKA/B and MCRT alone. Naloxone (2mg/kg) completely blocked the depressor effect of MCTR, but partially blocked that of EKA/B. The NO synthase inhibitor l-NAME (50mg/kg) partially blocked the depressor effects of EKA/B, MCRT, and EKA/B + MCRT. These results could help to better understand the role of tachykinin receptors, opioid receptors and neuropeptide FF receptors in cardiovascular system.